Let-7 microrna and mimetics thereof as therapeutics for cancer

ABSTRACT

The present invention relates to methods to treat or prevent cancers in a subject, in particular the present invention relates to a method of treating and/or preventing cancer comprising targeting cancer stem cells by administering miRNAs which have reduced expression or are lacking in the cancer stem cells. In some embodiments, the miRNAs that are reduced or lacking in cancer stem cells are let-7 miRNAs. In alternative embodiments, the present invention relates to a method of treating and/or preventing cancer comprising targeting cancer stem cells by administering miRNAs which have increased expression levels in the cancer stem cells. Another aspect of the present invention relates to methods to enrich for a cancer stem cell population. Another aspect of the present invention relates to methods to identify miRNAs which contribute to the self-renewal capacity of cancer stem cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/525,020, filed on Aug. 26, 2010, which is a 371 National Phase EntryApplication of International Application PCT/US2008/052654 filed Jan.31, 2008, which designated the U.S., and claims benefit under 35 U.S.C.§119(e) of U.S. Provisional Application Ser. No. 60/898,610 filed Jan.31, 2007, the contents of which is incorporated herein in its entiretyby reference.

GOVERNMENT SUPPORT

This invention was made with government support to ES under Contract No.30525022 awarded by the National Science Foundation of China, ContractNo. 2005CB724605 awarded by the 973 Program Project from Ministry ofScience and Technology of China.

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is2014-05-21_Sequence_Listing_(—)701039-059233-US.txt. The text file is76562 bytes and is being submitted electronically via EFS-Web,concurrent with the filing of the specification.

BACKGROUND OF THE INVENTION

Accumulating evidence suggests that many cancers are maintained in ahierarchical organization of rare, slowly dividing tumor-initiatingcells, rapidly dividing amplifying cells (precursor cells) anddifferentiated tumor cells^(1,2). Tumor-initiating (also termed cancerstem) cells have been identified in hematologic³⁻⁵, brain⁶⁻⁸,breast^(9,10), prostate¹¹ and colon cancers¹². Stem cells, which areself-renewing and can differentiate into heterogeneous cell populations,are highly tumorigenic^(1,2). Tumor-initiating cells are thought notonly to be the source of the tumor, but can also to be responsible fortumor progression¹³, metastasis^(14,15), resistance to cancer therapyand subsequent tumor recurrence^(16,17).

Although there is a growing consensus that cancer stem cells areimportant in generating tumors and for resistance to therapy andmetastasis, a major obstacle to their study is getting enough cellsbecause of their very low frequency in tumors^(9,10,12,37). Therefore,there is much need in the art for an efficient method for enriching forthese cancer stem cells.

In some organisms, miRNAs are known to play a role in maintainingstemness of embryonic stem (ES) cells, because ES cells deficient inmiRNA processing genes cannot be maintained²⁰. Previous studies haveshown an overall reduction in miRNA expression in embryonic or tissuestem cells²¹ and changes in specific miRNAs have been associated withself-renewal and differentiation of ES cells^(20,22). Moreover, miRNAexpression profiling has been shown to be useful for characterizing thestage, subtype and prognosis of some cancers^(18,23,24)

Based on the importance of cancers stem cells believed to be not only tobe the source of the tumor, but also to be responsible for tumorprogression¹³, metastasis^(14,15), resistance to cancer therapy andsubsequent tumor recurrence^(16,17), a method for reliably determiningif a subject has a cancer stem cell is needed in the art. In addition, amethod for reducing the occurrence of cancer stem cells and a method oftreating cancer stem cells in patients are also highly desirable.

SUMMARY OF THE INVENTION

The present invention relates to methods to treat or prevent cancers ina subject, in particular the present invention relates to a method oftreating and/or preventing cancer comprising targeting cancer stem cellsby administering miRNAs which have reduced expression or are lacking inthe cancer stem cells. In some embodiments, the miRNAs that are reducedor lacking in cancer stem cells are let-7 miRNAs. Conversely, inalternative embodiments, the present invention relates to a method oftreating and/or preventing cancer comprising targeting cancer stem cellsby administering agents which inhibit the expression of miRNAs, whichhave increased expression levels in the cancer stem cells. Anotheraspect of the present invention relates to methods to enrich for acancer stem cell population. Another aspect of the present inventionrelates to methods to identify miRNAs which contribute to theself-renewal capacity of cancer stem cells.

One aspect of the present invention relates to a method of treating orpreventing a cancer in a subject which comprises administering to thesubject a pharmaceutical composition comprising an effective amount ofat least one let-7 miRNA, or an agent that increases the expression of alet-7 miRNA, wherein the let-7 miRNA binds to and inhibits an RNAtranscript comprising a let-7 target sequence which is expressed in acancer stem cell. In some embodiments, the let-7 target sequencecomprises SEQ ID NO:9 or a homologue thereof. For example, the let-7target sequence that is a homologue of SEQ ID NO:9 can comprise SEQ IDNO: 10 or SEQ ID NO:11.

In some embodiments, the method comprises administering a pharmaceuticalcomposition comprising let-7 miRNA, where let-7 miRNA is encoded by alet-7-encoding nucleic acid construct. As a non-limiting example, thelet-7 miRNA can be a member of the let-7 family of miRNAs, such as, butnot limited to let-7a, let-7b, let-7c, let-7d, let-7e and let-7f andhomologues thereof that are effective in gene silencing. In someembodiments, the let-7 is let-7a or let-7a1.

In some embodiments, a let-7 miRNA can be a pri-miRNA, pre-miRNA, maturemiRNA or a fragment or variant thereof effective in gene silencing. Insome embodiments, the let-7 miRNA comprises SEQ ID NO:1 or a fragment orhomologue thereof effective in gene silencing. In alternativeembodiments, let-7 miRNA homologues can be used, for example the let-7miRNA from the let-7 miRNA family including, but not limited to, let-7miRNA comprising SEQ ID NOS:2-6 or a fragment or homologue thereofeffective in gene silencing. In some embodiments, a let-7 miRNA usefulin the methods disclosed herein is a pre-miRNA of SEQ ID NO:7 or afragment or homologue thereof effective in gene silencing. In someembodiments, a let-7 miRNA is a let-7a-1 stem-loop. In alternativeembodiments, the let-7 miRNA is let-7a-1 of SEQ ID NO:8 or a fragment orhomologue thereof effective in gene silencing. In other embodiments, thelet-7 miRNA is an RNA interference-inducing (RNAi) molecule including,but not limited to, a siRNA, dsRNA, stRNA, shRNA and gene silencingvariants thereof. In alternative embodiments the let-7 miRNA is an agentwhich binds and inhibits an RNA transcript comprising a let-7 targetsequence. Examples of such agents include, but are not limited to asmall molecule, protein, antibody, aptamer, ribozyme, nucleic acid ornucleic acid analogue.

In some embodiments, the methods as disclosed herein are useful for thetreatment or prevention of a cancer, for example where a cancer ischaracterized by the reduction or loss of a let-7 miRNA. In someembodiments the cancer comprises a cancer stem cell. In someembodiments, the cancer is a pre-cancer, and/or a malignant cancerand/or a therapy resistant cancer. In some embodiments, the cancer is abreast cancer.

In some embodiments, the let-7 miRNA or let-7 agent can further comprisea binding moiety and a targeting moiety, and in some embodiments thebinding moiety binds let-7 miRNA to the targeting moiety. In someembodiments, a targeting moiety is a cell surface receptor ligand orantigen-binding fragment thereof, for example a cell surface receptorligand including, but not limited to, CD133, CD44, mini-MUC; MUC-1;HER2/neu; HER2; mammoglobulin; labyrinthin; SCP-1; NY-ESO-1; SSX-2;N-terminal blocked soluble cytokeratin; 43 kD human cancer antigen;human tumor associated antigen (PRAT); human tumor associated antigen(TUAN); L6 antigen; carcinoembryonic antigen; CA15-3; oncoprotein18/stathmin (Op18); human glandular kallikrein (hK2); NY-BR antigens,tumor protein D52, and prostate-specific antigen; and early endosomeantigen 1 (EEA, c-kit, ABC7, SCA1 or combinations or antigen bindingfragments thereof. In some embodiments, a targeting moiety useful in themethods as disclosed herein is an antibody, for example an antibodyincluding not just complete or full length antibodies, but also antibodyderivatives, such as a single chain antibody, a Fab portion of anantibody or a (Fab′)₂ segment, which binds to a cell surface antigenpresent on a cancer cell. In some embodiments, a binding moiety usefulin the methods as disclosed herein is a protein or a nucleic acidbinding domain of a protein, and in some embodiments the binding moietyis fused to the carboxyl terminus of the targeting moiety, and in someembodiments, the binding moiety is the protein protamine or nucleic acidbinding fragment of protamine.

In some embodiments, the methods as disclosed herein further compriseadministering to the subject at least one or more additional cancertherapies, such as surgery, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy.

In some embodiments, the let-7 miRNA is administered to a subject morethan once, and can be administered before, after or at the same time asan additional cancer therapy or agent.

In some embodiments, the let-7 miRNA is encoded by a nucleic acid in avector, for example, a plasmid, cosmid, phagemid, or virus or variantsthereof, and in some embodiments the let-7 miRNA is operatively linkedto a promoter. In some embodiments, the vector further comprises one ormore in vivo expression elements for expression in human cells, such asa promoter or enhancer and combinations thereof.

In some embodiments, administration of the let-7 miRNA or let-7 agentscan be intravenous, intradermal, intramuscular, intraarterial,intralesional, percutaneous, subcutaneous, or by aerosol administration,or combinations thereof. In some embodiments, administration isprophylactic administration, and in alternative embodiments,administration is therapeutic administration.

In some embodiments, the methods and compositions as disclosed hereincan be administered to a subject, where the subject is, for example, amammal such as a human. In some embodiments, the subject has previouslyundergone at least one or more cancer therapies including, but notlimited to, surgery, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy.

Another aspect of the present invention relates to a method of treatingor preventing a cancer in a subject, the methods comprisingadministering to the subject an effective amount of at least one agentthat inhibits one or more genes and/or a product of such geneexpression, wherein the RNA transcript of (i.e. transcribed from) thegene comprises a let-7 target sequence, and the gene is gene silenced bylet-7 miRNA in non cancer cells.

In some embodiments, the let-7 target sequence comprises SEQ ID NO:9 ora homologue thereof effective in gene silencing. For example, the let-7target sequence can comprise SEQ ID NO:10 or SEQ ID NO:11 or a homologuethereof effective in directing gene silencing.

In some embodiments, the genes which comprise a let-7 target sequence intheir RNA transcript include, but are not limited to, RAS, lin-42, KRAS,GRB2, hbl-1, daf-12, pha-4 or human homologues thereof.

In some embodiments, an agent as disclosed herein can be, for example asmall molecule, nucleic acid, nucleic acid analogue, aptamer, ribozyme,peptide, protein, antibody, or variants and fragments thereof. In someembodiments, a nucleic acid agent can be DNA, RNA, nucleic acidanalogue, peptide nucleic acid (PNA), pseudo-complementary PNA (pcPNA),locked nucleic acid (LNA) or analogue thereof, and in embodiments wherethe nucleic acid agent is RNA, the RNA can be a small inhibitory RNA(RNAi), siRNA, microRNA, shRNA, miRNA and analogues and homologues andvariants thereof effective in gene silencing.

In some embodiments, a let-7 miRNA or agent can be administered to asubject via a variety of different routes, for example intravenous,intradermal, intramuscular, intraarterial, intralesional, percutaneous,subcutaneous, or by aerosol administration. In some embodiments,administration is prophylactic administration and in alternativeembodiments, administration is therapeutic administration. In someembodiments, the methods and compositions as disclosed herein can beadministered to a subject, where the subject is, for example, a mammalsuch as a human. In some embodiments, the subject has previouslyundergone at least one or more cancer therapies, such as, but notlimited to surgery, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy.

Another aspect of the present invention relates to a method to determineif a subject is at risk of having a metastasis or malignant cancer, themethod comprising assessing the presence of a let-7 miRNA in a testbiological sample obtained from the subject, wherein if the level of alet-7 miRNA in the test biological sample is reduced relative to thelevel of the let-7 miRNA in a reference sample, the subject is at riskof having a metastasis or a malignant cancer. In some embodiments, ifthe subject is identified as having a risk of metastasis or a malignantcancer, the method further comprises administering to the subject aneffective amount of a pharmaceutical composition comprising at least onelet-7 miRNA, or an agent that increases the expression of a let-7 miRNAaccording to claim 1.

In some embodiments, biological sample as disclosed herein is a tissuesamples, such as a tumor tissue sample or a cancer cell or tumor cell,for example a biopsy tissue sample obtained from the subject, such as abiopsy tissue sample is from a cancer. In some embodiments, the biopsysample is from breast cancer.

In some embodiments, let-7 miRNA levels can be determined by any methodsknown by persons of ordinary skill in the art. For example, let-7 miRNAlevels can be determined using a nucleic acid probe in, for example,Northern blot analysis, PCR, RT-PCR or quantitative RT-PCR. Examples ofa nucleic acid probe useful in the methods as disclosed herein include anucleic acid probe corresponding to SEQ ID NO:12 or SEQ ID NO:13 or SEQID NO:14 or a nucleic acid probe which specifically hybridizes to SEQ IDNO: 13 or SEQ ID NO:14, where such nucleic acid probes can be used inNorthern blot analysis, PCR, RT-PCR, quantitative RT-PCR and othermethods to determine expression levels of nucleic acids in a biologicalsample.

Another aspect of the present invention relates to a method to enrichfor cancer stem cells, the method comprising; (i) transplanting aplurality of cancer cells into a mammal, wherein the mammal isadministered a low dose cancer therapy, and allowing a sufficient periodof time for the cancer cells to form a tumor, (ii) removing the tumorfrom the mammal and dissociating the tumor into single cells, (iii)transplanting a plurality of the single cells into a mammal, wherein themammal is administered a low dose cancer therapy, and allowing asufficient period of time for the cancer cells to form a tumor, (iv)repeating steps (iii) and (iv) a plurality of times, for example, atleast 2 times and in some instances at least 3, 4, 5 or more times, (v)removing the tumor from the mammal and dissociating the tumor intosingle cells, and (vi) culturing the cells as single-cells forsufficient time to form an embryoid body, wherein the embryoid bodycomprise a population of cells enriched in cancer stem cells.

In some embodiments of methods to enrich for cancer stem cell, theembryoid body is a mammosphere. In some embodiments, a cancer cell thatis transplanted into a mammal is a primary cancer cell or a cancer cellline, such as a genetically modified primary cancer cell or cancer cellline. In some embodiments, a cancer cell is from a biological sample,such as a biopsy tissue, for example a cancer biopsy tissue sample.

In alternative embodiments where the cancer cell is of a cancer cellline, the cancer cell line can be derived from a tumor or cancerincluding, but not limited to, breast cancer, lung cancer, head and neckcancer, bladder cancer, stomach cancer, cancer of the nervous system,bone cancer, bone marrow cancer, brain cancer, colon cancer, esophagealcancer, endometrial cancer, gastrointestinal cancer, genital-urinarycancer, stomach cancer, lymphomas, melanoma, glioma, bladder cancer,pancreatic cancer, gum cancer, kidney cancer, retinal cancer, livercancer, nasopharynx cancer, ovarian cancer, oral cancers, bladdercancer, hematological neoplasms, follicular lymphoma, cervical cancer,multiple myeloma, osteosarcomas, thyroid cancer, prostate cancer, coloncancer, prostate cancer, skin cancer, stomach cancer, testis cancer,tongue cancer, or uterine cancer. In some embodiments, the cancer cellline is a breast cancer cell line.

In some embodiments, cancer cells are transplanted into a mammal whichcan be any mammal, such as a monkey, rodent or genetically modifiedrodent. In some embodiments, the mammal is an immunocompromised mammal,for example, a NOD/SCID mouse.

In some embodiments of methods to enrich for cancer stem cells, a cancertherapy can be for example, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy. An example ofchemotherapy is the chemotherapy agent Epirubicin.

In some embodiments of methods to enrich for cancer stem cells,administration of the low-dose cancer therapy can be continuousadministration or in alternative embodiments, non-continuousadministration, for example twice a day, once a day, every other day,twice a week, once a week, every other week or once a month. In someembodiments, administration can be by any route known by persons ofordinary skill in the art, such as intravenous, intradermal,intramuscular, intraarterial, intralesional, percutaneous, subcutaneous,intraperitoneal, or aerosol administration. In some embodiments, thetime to culture the cells to enable them to form an embryoid body is asufficient period of time is a period of time to allow the tumors toreach at least about 2 cm in diameter. In some embodiments,transplanting the cells relates to transplanting the cells into themammary fat pad of a female rodent.

Another aspect of the present invention relates to methods to identifymiRNAs that contribute to the self-renewal capacity of cancer stemcells, the method comprising obtaining cancer stem cells, for example bythe methods as disclosed herein, and analyzing the expression of aplurality of miRNAs from said cancer stem cells, and comparing theexpression profile of miRNAs of said cancer stem cells with the miRNAexpression profile from a reference sample, wherein an increased orreduced level of expression of an miRNA in the cancer stem cells ascompared to the level of miRNA in the reference sample identifies anmiRNA that contributes to the self-renewal capacity of the cancer stemcells. In some embodiments, a reference sample useful in the methods asdisclosed herein is a non-stem cell cancer cell, or a differentiatedcancer stem cell.

Any means to analyze a miRNA expression profile known by persons ofordinary skill in the art can be used in the methods as disclosedherein, for example by microarray assay.

In some embodiments of the methods as disclosed herein, the methods canfurther comprise assessing the miRNA that contributes to theself-renewal capacity, the method comprising introducing into the cancerstem cell the miRNA if the miRNA is identified to be expressed at alower level in a cancer stem cell as compared to the reference sample,and assessing the ability of the cancer stem cell to from a embryoidbody, wherein a reduced ability to from a embryoid body indicates thatthe miRNA contributes to a cancer stem cell's self-renewal capacity. Inalternative embodiments, the methods can further comprise assessing themiRNA that contributes to the self-renewal capacity, the methodcomprising inhibiting the expression of an miRNA in the cancer stem cellif the miRNA is identified to be expressed at a higher level in thecancer stem cell as compared to the reference sample, and assessing theability of the cancer stem cell to from an embryoid body, wherein areduced ability to from an embryoid body identified an miRNA thatcontributes to cancer stem cell self-renewal capacity.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a let-7 miRNA and a pharmaceutically acceptablecarrier, wherein the let-7 miRNA binds to and inhibits an RNA transcriptcomprising a let-7 target sequence.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising an agent which increases the expression of alet-7 miRNA and a pharmaceutically acceptable carrier, wherein the let-7miRNA binds to and inhibits an RNA transcript comprising a let-7 targetsequence.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising an agent which decreases the expression of atleast one of miR-129, miR-140, miR-184 or miR-198 and a pharmaceuticallyacceptable carrier.

In some embodiments, a pharmaceutical composition as disclosed hereincomprises a let-7 miRNA or an agent which increases a let-7 miRNA, andthe let-7 target sequence comprises SEQ ID NO:9 or a homologue thereofeffective in gene silencing, such as a let-7 target sequence comprisingSEQ ID NO: 10 or SEQ ID NO:11.

In some embodiments, the pharmaceutical compositions as disclosed hereinare useful of for the treatment or prevention of cancer in a subject,for example, for the treatment or prevention of breast cancer. In someembodiments, the subject is a mammal, such as a human subject.

In some embodiments where a pharmaceutical composition comprises anagent, the agent can be, for example, a small molecule, nucleic acid,nucleic acid analogue, aptamer, ribozyme, peptide, protein, antibody, orvariants and fragments thereof. In some embodiments where the agent is anucleic acid, the nucleic acid can be for example, DNA, RNA, nucleicacid analogue, peptide nucleic acid (PNA), pseudo-complementary PNA(pcPNA), locked nucleic acid (LNA) or analogue thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1G show that breast cancer cells under pressure of chemotherapyare enriched for breast tumor-initiating cells. FIG. 1A shows thatprimary breast cancer cells isolated from surgical specimens frompatients who received preoperative neoadjuvant chemotherapy aresubstantially enriched for self-renewing cells with the properties ofcancer stem cells, compared to untreated patients. Representative imagesshow increased numbers and size of mammospheres after 15 d of culture,and FIG. 1B shows a higher percentage of CD44⁺CD24⁻ cells in freshlyisolated tumor cells from patients who received chemotherapy. Similarly,FIGS. 1C-1G show that passaging the human breast cancer cell line SKBR3in Epirubucin-treated immunodeficient mice enriches for tumor-initiatingcells. SK-3rd cells dissociated from the 3^(rd) passage xenograft areself-renewing. They have enhanced ability, compared to the parentalline, to form mammospheres, and the mammospheres can be repetitivelypassaged in vitro and are larger. FIG. 1C shows numbers of primary,secondary (generated from dissociated primary mammospheres) and tertiary(generated from dissociated secondary mammospheres) mammospheres on day15 from 1000 cells. *, P<0.001 compared with SKBR3. FIG. 1D showsmammospheres generated from single-cell cultures of SK-3rd and SKBR3,imaged on indicated days of suspension culture. Shown are the mean±SDnumber of cells/sphere for each timepoint. *, P<0.001 compared withSKBR3. FIGS. 1F and 1G show that when SK-3rd cells have the phenotype ofbreast tumor-initiating cells; they are CD44⁺CD24⁻Oct4⁺. FIG. 1E (left)shows when SK-3rd mammospheres are dissociated, and removed from growthfactors and plated on collagen, they adhere and differentiate (FIG. 1E,right) and assume the parental SBKR3 phenotype (Oct-4 immunoblot, asshown in FIG. 1F). FIG. 1G shows that SK-3rd and SKBR3 cells cultured asspheres are CD44⁺CD24⁻. When they differentiate in adherent cultures,they gradually assume the parental SBKR3 phenotype, but somewhat morerapidly for SKBR3 mammospheres.

FIGS. 2A-2G show that SK-3rd cells and primary breast cancer cells fromchemotherapy-treated patients have low expression of let-7 familymiRNAs. FIG. 2A shows miRNA array analysis, which shows that miRNAs aredifferentially expressed in SK-3rd cells cultured in mammospheres (1) oradhered for 8 hr (2), 24 hr (3), or 10 days (4) and parent SKBR3 (5).Most miRNAs, including all let-7 homologs or let-7 family members, arereduced in SK-3rd cultured in mammospheres or just adhered for 8 hr, andincrease during differentiation to similar levels as SKBR3. FIG. 2Bshows the microarray results for let-7 were verified by Northern blotusing a nonspecific let-7 probe, and FIG. 2C shows verification byqRT-PCR amplified for let-7a (mean±SD relative to U6). FIG. 1D showsthat infection of SK-3rd with lentivirus expressing pre-let-7a(lenti-let-7) vs. empty vector increased let-7 expression to levelscomparable to differentiated SK-3rd. let-7 function, assayed byluciferase assay in cells transfected with a reporter gene containing alet-7 target site in its 3′-UTR, is negligible in SK-3rd cells butincreases upon differentiation or by infection with lenti-let-7 (*,P<0.001 compared with SK-3rd). Transfection with let-7 ASO reducesendogenous or exogenous let-7 activity (#, P<0.01 compared to cells nottransfected with let-7 ASO). FIG. 2E shows that H-RAS, a target oflet-7, is highly expressed in SK-3rd, but not in the differentiatedadherent cell line or SKBR3 (protein assayed by immunoblot relative toα-actin). FIGS. 2F and 2G shows that infection with lenti-let-7 orlentivirus encoding RAS-shRNA, but not GFP-shRNA or empty vector,suppresses H-RAS expression in SK-3rd cells, while transfection of SKBR3with let-7 ASO augments H-RAS protein. FIG. 2F shows let-7 is alsoreduced in primary mammospheres from isolated tumor cells from patientswho received neoadjuvant chemotherapy, compared to patients who did not.FIG. 2F shows a northern blot of representative samples probed for let-7family miRNAs and FIG. 2G shows qRT-PCR results for 5chemotherapy-treated patients and 6 untreated patients amplified forlet-7a (mean±SD relative to U6). In FIG. 2F, samples are mammospheric(lane 1) and adherent differentiated cell (lane 2) RNA fromrepresentative chemotherapy patient, compared with RNA extracted from apatient who did not receive chemotherapy (lane 3).

FIGS. 3A-3F shows that SK-3rd cells engineered to express let-7a lose“stemness”. FIG. 3A shows that single cell cultures of dissociatedSK-3rd cells, infected with lenti-let-7 or lentivirus expressingRAS-shRNA, but not GFP-shRNA or empty vector, form fewer mammospheres,and FIG. 3B shows that mammospheres that do form develop more slowly andare reduced in cell number (*, P<0.0001; compared to untransducedcells). Conversely, FIG. 3C shows that SKBR3 cells transfected withlet-7a ASO, but not control lin-4 ASO, generate 10-fold moremammospheres. FIG. 3D shows that let-7 expression, assayed by qRT-PCRrelative to U6, increases during in vitro differentiation of SK-3rd.FIG. 3E shows that SK-3rd cells infected with lenti-let-7, and to alesser extent lentivirus expressing RAS-shRNA, proliferate less duringin vitro differentiation than untransduced or control transduced cellsas measured by [³H]-incorporation *, P<0.01; #, P<0.05 compared withuntransduced SK-3rd. FIG. 3F shows that after 10 d of in vitrodifferentiation, SK-3rd cells over-expressing let-7a, but not RAS-shRNA,have half as many undifferentiated cells lacking expression of thecytokeratins CK14 or CK18.

FIGS. 4A-4D shows that expression of pre-let-7a by SK-3rd cellssuppresses tumor xenograft growth in NOD/SCID mice. FIG. 4A shows tumorvolume which was measured after subcutaneous mammary fat pad inoculationof 2×10³ (top), 2×10⁴ (middle) or 2×10⁵ (bottom) SKBR3 cells or SK-3rdcells that were untransduced or transduced with empty vector or toexpress let-7. The number in the figure legend indicates the number ofmice who developed tumors. 10 mice were in each group. Over-expressionof let-7a led to fewer tumors and the tumors that arose grew moreslowly. FIG. 4B shows tumors that grew in mice inoculated with 2×10⁵cells had similar histology by hematoxylin and eosin staining (HE,magnification 200×), but the SK-3rd tumors, either untransduced ortransduced with empty vector, had higher expression of H-RAS (400×, andshown in FIG. 4C) and a higher proliferative index assessed by PCNAstaining (400×, shown in FIG. 4D), than the parental SKBR3 cells orSK-3rd cells transduced with lenti-let-7.

FIGS. 5A-5C show that SK-3rd cells transduced with pre-let-7a are lesslikely to metastasize. FIG. 5A shows Hematoxylin and eosin staining ofthe lung (×200) and liver (×400) of mice implanted subcutaneously with2×10⁵ SK-3rd cells (either untransduced or transduced with lentivirusvector or lenti-let-7) or SKBR3. Arrows indicate focal metastasis. FIG.5B shows mean±SD wet lung weight in mice bearing tumor xenografts(n=10/group). FIG. 5C shows the expression of human HPRT mRNA relativeto mouse GAPDH, by qRT-PCR. The numbers indicate the number of animalsin each group of 10 with lung or liver metastasis. N.D., not detected

FIG. 6 shows that SK-3rd cells in single cell suspension cultures haveenhanced capability to form mammospheres. Nonadherent mammospheresgenerated from single-cell suspension cultures of SK-3rd and SKBR3 cellswere counted for 20 d of culture. *, P<0.001 SK-3rd compared with SKBR3cells at the same time point.

FIGS. 7A-7D shows sequences of let-7 miRNA. FIG. 7A shows the nucleicacid sequences of isoforms or homologues of let-7, let-7a (SEQ ID NO:1);let-7b (SEQ ID NO:2); hsa-let-7c (SEQ ID NO:3); hsa-let-7d (SEQ IDNO:4); hsa-let-7e (SEQ ID NO:5); hsa-let-7f (SEQ ID NO:6). FIG. 7B showsthe nucleic acid sequence of let-7 miRNA (SEQ ID NO:7). FIG. 7C showsthe nucleic acid sequence homo sapiens let-7a1 stem loop (SEQ ID NO: 8).FIG. 7D shows the nucleic acid sequence of the let-7 target sequence(5′-AACTATACAACCTACTACCTCA-3′; SEQ ID NO: 9) and 2 let-7 targetsequences (SEQ ID NO:10 and SEQ ID NO:11) inserted into the reportervector.

FIGS. 8A-C show that breast cancer cells under pressure of chemotherapyare enriched for tumor initiating breast cancer cells (BT-IC). FIG. 8Ashows that the majority of freshly isolated SK-3rd cells are CD44+CD24−,as expected for BT-IC, while cells with this phenotype are rare in SKBR3(representative data of five experiments shown). FIG. 8B shows that whenSK-3rd spheres are dissociated, removed from growth factors, and platedon collagen for 8 hr (top), they do not express luminal (Muc1 and CK-18)or myoepithelial (CK-14 and α-SMA) differentiation markers, while afterfurther differentiation (bottom), they develop into elongated cells withsubpopulations staining for either differentiated subtype. FIG. 8C showsthat freshly isolated SK-3rd cells are enriched for Hoechst low SP cellscompared with SKBR3 cells.

FIGS. 9A-9F show that let-7 miRNA is reduced in mamospheric SK-3^(rd)cells in primary tumor initiating breast cancer cells (BT-IC). FIG. 9Ashows Northern Blot probed for let-7m and FIG. 9B shows results fromqRT-PCR amplified for let-9a (mean±SD relative to U6) to verify themicroarray results. Spheres derived from either SK-3rd or SKBR3 showsimilar low expression of let-7 that increases gradually beginning 1days following induction of differentiation and plateaus within 6 days.#, p<0.01; *, p<0.001 as compared with cells cultured in spheres. Errorbars correspond to mean±SD. FIG. 9C shows HMGA2, a target of let-7, ishighly expressed in mammospheric SK-3rd but not in differentiatedadherent SK-3rd or SKBR3 (protein assayed by immunoblot relative tob-actin). Infection with lenti-let-7 or lentivirus encoding RAS- orHMGA2-shRNA, but not GFP shRNA or vector, suppresses HMGA2 expression,respectively, in mammospheric SK-3rd cells, while transfection of SKBR3with let-7 ASO augments HMGA2 protein. In addition, FIGS. 9D-F showtumors from eight untreated patients and five patients treated withneoadjuvant chemotherapy were enriched for BT-IC by sorting forlin⁻CD44⁺CD24⁻ cells or by growth as mammospheres. Tumors depleted ofBT-IC by adherent growth or by excluding CD44⁺CD24⁻ cells also havereduced let-7 compared to adjacent normal breast tissue. FIG. 9D showsFACS analysis and FIG. 9E shows Northern blots probed for let-7 and U6for representative untreated (#7), and neoadjuvant chemotherapy treated(#5) patients. FIG. 9F shows mean±SD of relative let-7 expression forall samples analyzed by qRT-PCR. Infection with lenti-let-7 increaseslet-7 in BT-IC-enriched primary cells. #, p<0.05; *, p<0.01 comparedwith samples depleted of CD44⁺CD24⁻ cells.

FIG. 10A-10D show that silencing HMGA2 reduces the undifferentiatedsubpopulation and proliferation of SK-3rd cells but does notsignificantly alter mammosphere formation. FIG. 10A shows thatBT-IC-enriched cells, sorted for lin⁻CD44⁺CD24^(−/low) phenotype fromprimary chemotherapy-naive breast tumors, have a markedly highercapacity to form mammospheres compared with CD44⁺CD24⁻-depleted cells.Transduction with lenti-let-7, but not lentivector, reduces mammospheregeneration. *, p<0.001 compared with untransduced cells. Mammosphereformation by let-7-transduced BT-IC is also significantly reduced onserial passage but is stable in untransduced cells. FIG. 10B showssingle-cell cultures of dissociated SK-3^(rd) cells, infected withlenti-HMGA2-shRNA, form a comparable number of mammospheres asuninfected cells or cells infected with lenti-GFP-shRNA or lentivector.Lenti-let-7 was used as a positive control. *, p<0.01 as compared withuntransduced SK-3rd. FIG. 10C shows that silencing HMGA2 withlenti-HMGA2-shRNA reduces proliferation of SK-3rd cells on day 4 of invitro differentiation in adherent cultures (peak of proliferation), butnot as much as lenti-let-7 transduction. Cell proliferation was measuredby [3H]-incorporation *, p<0.01; #, p<0.05 compared with untransducedSK-3rd. FIG. 10D shows that transduction with lenti-HMGA2-shRNA orlenti-let-7, but not with lenti-GFP-shRNA or vector, similarly reducesthe proportion of lin⁻ cells in SK-3rd cells cultured in mammospheres.*, p<0.01 compared with vector transduced cells. Error bars correspondto mean±SD.

DESCRIPTION OF THE INVENTION

In accordance with the present invention, the inventors have discoveredthat preoperative chemotherapy in patients enriches for tumor-initiatingcells, herein referred to as “cancer stem cells”. The inventors havealso discovered that cancer stem cells lack or have reduced expressionof specific miRNA as compared with normal tissue and non-stem cellcancer cells. The inventors herein have discovered cancer stem cellslack or have reduced miRNA expression of miRNAs including, but notlimited to, let-7, miR-107, miR-10a, miR-128a, miR128b, miR-132,miR-138, miR-16, miR-17, miR-195, miR-199a, miR-20, miR-200a, miR-200b,miR-200c, miR-20b, miR-22. In particular, the inventors have discoveredthat cancer stem cells have reduced or lack let-7 miRNA expressioncompared with normal tissue and non-stem cell cancer cells. Furthermore,the inventors have discovered that breast cancer stem cells have reducedexpression of let-7 which is not the case in non-stem cell breast cancercells, and that reduced expression or lack of let-7 is required tomaintain “stemness” of the cancer stem cells, for example lack of let-7enables cancer stem cells to self-renew and be maintained in anundifferentiated state. The inventors also discovered that cancer stemcells that lack or have reduced expression of let-7 as compared tonon-stem cell cancers were highly malignant and were more likely toresult in metastasis in the liver and lung. Thus, the inventors havediscovered that cells having reduced expression or lacking specificmiRNAs, for example but not limited to, cells having reduced expressionor lacking expression of let-7 miRNA identifies a cell as a cancer stemcell and identifies the cell as contributing to increased tumorigenicityand cancer metastasis.

In another aspect of the invention, the inventors discovered thatexpression of specific miRNAs is reduced or absent in cancer stem cells,and that expression of such miRNAs reduces these cancer stem cells'self-proliferative capacity and converts the cancer stem cells fromhighly malignant and metastasizing cancer stem cells into less malignantcells. For example, the expression of let-7 in cancer stem cells wasdiscovered to reduce the cells capacity for self-proliferation andrender them less malignant. Thus, the inventors have discovered thatspecific miRNA that are reduced and/or lacking in cancer stem cells actas tumor suppressors; for example, let-7 acts as a tumor suppressor.

Another aspect of the invention relates to the inventors discovery of anovel method to enrich for cancer stem cells using repeated passaging ofcancer cells. The inventors demonstrate a method for enriching for apopulation of cancer stem cells by (i) transplanting cancer cells in ananimal model in vivo, and allowing the cancer cells to grow into a tumorin the presence of low dose chemotherapy, then harvesting the cancercells from the tumor and (ii) re-transplanting the harvested cancercells into a subsequent animal model and repeating step (i). The steps(i) and (ii) can be repeated a number of times, for example at least 2,or at least 3 or at least 4 or up to as many as 10 or more times toenrich for a population of cancer stem cells that are resistant to atleast one or more different low dose chemotherapy agents.

Further, the inventors have discovered a method to identify miRNAs thatcontribute to cancer stem cells' self-proliferative capability and“stemness”. As disclosed herein, the term “stemness” is defined below,and typically refers to the self-proliferative capacity of an immatureor non-terminally differentiated cell, for example, the capacity of acell to produce a daughter cell, which themselves can be induced toproliferate and produce progeny that subsequently differentiate into oneor more mature cell types, while also retaining one or more cells withparental developmental potential.

Accordingly, the present invention provides methods to treat cancers bytargeting cancer stem cells, the method comprising targeting the cancerstem cell with miRNAs or mimetics thereof to increase the levels ofmiRNAs which are reduced or lacking in cancer stem cells as compared tonon-stem cell cancer cells. As a non-limiting example, the presentinvention provides methods to treat and prevent cancers by targetingcancer stem cells with let-7 miRNA, and/or a mimetic thereof, toincrease levels of let-7 miRNA in the cancer stem cell. An increase inlet-7 miRNA in the cell will increase let-7 mediated gene silencing inthe cancer stem cell, which will cause a reduction in the self-renewaland proliferative capacity and/or differentiation capacity of a cancerstem cell to a less malignant cell. In some embodiments, any form oflet-7 can be used in the methods as disclosed herein, for example anynucleic acid or any agent which has a minimum biological activity ofbinding to and inhibiting the let-7 target sequence5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9). In some embodiments, onecan use let-7a, and in alternative embodiments, let-7 miRNA can be inthe form of any of the following, but not limited to, let-7 pre-miRNA,let-7 pri-miRNA or mature let-7 miRNA or homologues, fragments andvariants thereof that retain a gene regulatory biological activity ofthe mature let-7 miRNA, especially the ability to downregulate theexpression of a target gene by miRNA-mediated gene silencing.

In alternative embodiments, cancers can be treated by targeting thecancer stem cell with any one or a combination of the following miRNAsand mimetics thereof: miR-107; miR-10a; miR-128a; miR128b; miR-132;miR-138; miR-16; miR-17; miR-195; miR-199a; miR-20; miR-200a; miR-200b;miR-200c; miR-20b and miR-22.

In some embodiments, the invention provides methods to treat cancers bytargeting cancer cells with a plurality of different miRNA and/or miRNAmimetics to increase the levels of more than one miRNA that are reducedor lacking in the cancer stem cells compared with non-stem cell cancercells.

Conversely, the inventors have also discovered that cancer stem cellshave increased expression of other specific miRNAs as compared withnormal tissue and non-stem cell cancer cells. For example, the inventorshave discovered that cancer stem cells have an increased level ofexpression of at least the following miRNA's: miRNAs miR-129; miR-140;miR-184; and miR-198. Accordingly, the present invention also providesmethods to treat cancers by targeting cancer stem cells, the methodcomprising targeting the cancer stem cell with agents which inhibit theexpression of miRNAs which are increased or elevated in the cancer stemcells as compared with non-stem cell cancer cells. As a non-limitingexample, the invention provides methods to treat cancers by targetingcancer stem cells with an agent that inhibits the activity and/orexpression of miR-198 to reduce levels of miR-198 miRNA in the cancerstem cell. Such an agent can be an inhibitory nucleic acid molecule, forexample but not limited to antisense nucleic acid molecules andRNA-interference molecules such as siRNA, etc.

Therefore in some embodiments, the methods of the present inventionrelate to the treatment of cancers by targeting cancer stem cells, themethod comprising upregulating miRNAs that are reduced or lacking incancer stem cells. As a non-limiting example, let-7 miRNAs can beupregulated by providing, pharmaceutical compositions comprising let-7miRNA and/or let-7 mimetics to a cancer stem cell in a therapeuticallyeffective amount for the treatment of cancer. In such embodiments, thecancer comprises a cancer stem cell and/or a cell with reduced orlacking let-7 expression.

In some embodiments, the pharmaceutical composition comprising miRNA ormimetics thereof, for example let-7 and/or let-7 mimetics isadministered with, at the same time, or sequential to another agent ortherapy, for example a cancer therapy. Such additional agents include,but are not limited to, surgery, chemotherapy, radiotherapy,thermotherapy, immunotherapy, hormone therapy and laser therapy.

Another aspect of the invention relates to use of let-7 miRNA and/orlet-7 mimetics as diagnostics and as therapeutics. In some embodiments,let-7 miRNAs and/or let-7 mimetics are administered to a subject in apharmaceutical composition where the subject has a cancer stem cell. Theadministration can be a treatment and/or prophylaxis for cancer, wherethe subject has at least one cancer stem cell. The subject can have, ornot have, symptoms or manifestation of cancer, since cancer stem cellscan exist in the absence of symptoms of cancer. In some embodiments, thepharmaceutical compositions comprising let-7 miRNA and/or let-7 mimeticsare administered to a subject with a cancer stem cell.

The cancer stem cell can be present in any type of cancer including, forexample breast cancer. In some embodiments, the cancer is atreatment-resistant cancer, for example, but not limited to a cancerwhich is resistant to chemotherapy, such as a chemotherapy-resistantbreast cancer.

In another aspect of the present invention, methods are provided totreat cancers by targeting the cancer stem cells, the method comprisingtargeting the cancer stem cell with agents that inhibit genes and/ortheir gene products (i.e. mRNAs or proteins) which are normally genesilenced by the miRNAs that are reduced in the cancer stem cells, forexample, genes that are gene silenced by let-7 miRNA. Such genes thatare regulated by miRNA are genes which comprise miRNA target sequencesin their mRNA. For example, genes silenced by let-7 comprise a let-7target sequence within their mRNA. The let-7 target sequence can be inthe 5′UTR, 3′UTR or coding sequence. Examples of such genes thatcomprise a let-7 target sequence in their mRNA include, but are notlimited to, RAS, HRAS, KRAS, lin-42, GRB2, hbl-1, daf-12 and pha-4, orhuman homologues thereof. In some embodiments, agents inhibit theactivity and/or the expression of genes that are gene silenced by miRNAsthat are reduced or lacking on cancer stem cells. As a non-limitingexample, in some embodiments an agent inhibits the activity and/orexpression of genes comprising let-7 target sequence within their mRNA.

In some embodiments, the methods of the present intervention relate tothe treatment of cancers by targeting cancer stem cells, the methodcomprising administering a pharmaceutical composition comprising atleast one agent that inhibits the activity and/or the expression of atleast one gene that is gene silenced by miRNA that are reduced orlacking in cancer stem cells. As a non-limiting example, apharmaceutical composition comprising at least one agent that inhibitsthe activity and/or the expression of at least one gene that is genesilenced by let-7 and/or comprises a let-7 target within their mRNA isadministered to a cancer stem cell in a therapeutically effective amountfor the treatment of cancer. In such embodiments, the cancer comprises acancer stem cell and/or a cell with reduced or lacking let-7 expression.

In another aspect of the present invention, methods for diagnosingwhether a subject is at risk of having or has a metastasis or amalignant cancer are provided. In some embodiments, the methods compriseassessing the level of let-7 in a biological sample from the subject,and if the level of let-7 is below a reference level, the subject isidentified as being at risk of having a metastasis and/or malignantcancer. In some embodiments, the biological sample is from a cancerbiopsy. In some embodiments, the cancer biopsy is a breast cancerbiopsy. In such embodiments, if the level of let-7 in the biologicalsample obtained from the subject is below the reference level, thesubject is administered a pharmaceutical compositions comprising let-7miRNA and/or a let-7 mimetic.

In another embodiment, the cancer to be treated is any cancer thatcomprises cancer stem cells. In alternative embodiments, the cancer tobe treated is any cancer characterized by reduced expression and/or lackof let-7 miRNA expression. In some embodiments, the cancer is breastcancer. In some embodiments, the cancer is a resistant cancer, forexample a multi-drug and/or chemotherapy-resistant cancer. In someembodiments, the cancer is lung or colon cancer.

In some embodiments, the let-7 miRNA are nucleic acids, include but notlimited to let-7 pri miRNA, let-7 pre-miRNA, mature let-7 miRNA orhomologues, fragments or variants thereof that retain the biologicalactivity of the mature let-7 miRNA.

In alternative embodiments, mimetics of let-7 miRNA are useful in themethods of the present invention. A let-7 mimetic is an entity or agentthat functions as a let-7 miRNA, for example, a nucleic acid or agentwhich has a minimum biological activity of binding to and inhibitingexpression of a gene comprising the let-7 target sequence5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9). Examples of let-7 mimeticsinclude, but are not limited to, small molecules, proteins, nucleicacids, ribosomes, aptamers, antibodies and nucleic acid analogues thatmimic let-7 miRNA. Nucleic acid let-7 mimetics can also include, but arenot limited to, RNA interference-inducing molecules (RNAi), includingbut not limited to, siRNA, dsRNA, stRNA, shRNA and modified versionsthereof, where the RNA interference (RNAi) molecule has a minimumbiological activity of binding to and inhibiting the expression of agene comprising the let-7 target sequence 5′-AACTATACAACCTACTACCTCA-3′(SEQ ID NO: 9).

Effective, safe dosages can be experimentally determined in modelorganisms and in human trials by methods well known to one of ordinaryskill in the art. The let-7 miRNA and/or let-7 mimetics in apharmaceutical composition can be administered alone or in combinationwith adjuvant cancer therapy such as surgery, chemotherapy,radiotherapy, thermotherapy, immunotherapy, hormone therapy and lasertherapy, to provide a beneficial effect, e.g. reduce tumor size, reducecell proliferation of the tumor, inhibit angiogenesis, inhibitmetastasis, or otherwise improve at least one symptom or manifestationof the disease.

Another aspect of the invention provides methods for the enrichment ofcancer stem cells. The method relates to sequential passaging of cancercells in vivo when exposed to low dose chemotherapy.

In another embodiment, the present invention provides methods toidentify miRNAs that contribute to the self-proliferative capacityand/or tumorogenicity of a cancer stem cell. In some embodiments, themethod comprises comparing the miRNA expression profile of a cancer stemcell enriched by the methods as disclosed herein, with the miRNAexpression profile of a reference sample, such as, but not limiting toan expression profile of a non-stem cancer cell. A change in a miRNA inthe cancer stem cell as compared with a reference sample identifies amiRNA that contributes, in whole or in part, to the self-proliferativecapacity and/or tumorogenicity of the cancer stem cell. In furtherembodiments, the present invention provides methods to assess the roleand level of such contribution of the identified miRNA to the cancerstem cells self-proliferative capability, the method comprising eitherintroducing or inhibiting the miRNA in the cancer stem cell, dependingupon whether the miRNA being assessed is downregulated or upregulatedrespectively, in the cancer stem cell as compared with the referencesample.

DEFINITIONS

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, the term “let-7” refers to a nucleic acid or an agentwhich has a minimum biological activity of binding to (or hybridizingto) and inhibiting the expression of a gene comprising the let-7 targetsequence, where the target sequence is 5′-AACTATACAACCTACTACCTCA-3′ (SEQID NO: 9). A let-7 can be assessed for its ability to bind to andinhibit the target sequence SEQ ID NO:9 using the let-7 luciferase assayas disclosed herein in the Examples, for example by using thepMIR-REPORT™ luciferase reporter vector with a let-7 target sequence(SEQ ID NO: 9) (as well as SEQ ID NO:10 and SEQ ID NO:11) cloned intoits 3′UTR. let-7 can refer to a nucleic acid encoding a let-7 miRNAcorresponding to SEQ ID NO: 1. “let-7” also refers to a nucleic acidencoding a let-7 miRNA or homologues and variants of SEQ ID NO: 1,including conservative substitutions, additions, and deletions thereinwhich do not adversely affect the structure or function, and where suchhomologues and variants have the same function or same activity of let-7encoded by SEQ ID NO:1 and are capable of binding to and inhibiting theexpression of a gene comprising the let-7 target sequence5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9). Preferably, let-7 refers tothe nucleic acid encoding let-7 from C. elegans (NCBI Accession No.AY390762), and in some embodiments, let-7 refers to the nucleic acidencoding a let-7 family member from humans, including but not limitedto, NCBI Accession Nos. AJ421724, AJ421725, AJ421726, AJ421727,AJ421728, AJ421729, AJ421730, AJ421731, AJ421732, and functional orbiologically active sequence variants of let-7, including alleles, andin vitro generated derivatives of let-7 that demonstrate let-7 activity.The term “let-7 family” includes let-7 homologues and isoforms, forexample but not limited to let-7 family members including let-7a (SEQ IDNO:1); let-7b (SEQ ID NO:2); hsa-let-7c (SEQ ID NO:3); hsa-let-7d (SEQID NO:4); hsa-let-7e (SEQ ID NO:5); hsa-let-7f (SEQ ID NO:6).

A let-7 agent as also referred to herein also encompasses a “let-7mimetic” and means an agent which binds to and inhibits the let-7 targetsequence 5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9). In this context, alet-7 agent can be any agent or RNA interference-inducing molecule, forexample but not limited to unmodified and modified double stranded (ds)RNA molecules including, short-temporal RNA (stRNA), small interferingRNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA),double-stranded RNA (dsRNA). Alternatively, a let-7 agent can be a smallmolecule, protein, aptamer, nucleic acid analogue, antibody etc. thatbinds to and inhibits the let-7 target sequence SEQ ID NO:9; Activity ofa let-7 agent can be assessed using, for example, the let-7 luciferaseassay as disclosed herein in the Examples which uses the pMIR-REPORT™luciferase reporter vector with a let-7 target sequence (SEQ ID NO: 9)cloned into its 3′UTR.

The terms “same activity” or “same function” as used in reference to thesame activity or function of let-7 means a let-7 molecule which can bindto and inhibit the target sequence SEQ ID NO:9 with at least 80% of theefficiency, or greater efficiency, as the wild type let-7 (SEQ ID NO:1),as assessed using, for example, the let-7 luciferase assay as disclosedherein in the Examples.

The terms “microRNA” or “miRNA” used interchangeably herein, areendogenous RNAs, some of which are known to regulate the expression ofprotein-coding genes at the posttranscriptional level. As used herein,the term “microRNA” refers to any type of micro-interfering RNA,including but not limited to, endogenous microRNA and artificialmicroRNA. Typically, endogenous microRNA are small RNAs encoded in thegenome which are capable of modulating the productive utilization ofmRNA. A mature miRNA is a single-stranded RNA molecule of about 21-23nucleotides in length which is complementary to a target sequence, andhybridizes to the target RNA sequence to inhibit expression of a genewhich encodes a miRNA target sequence. miRNAs themselves are encoded bygenes that are transcribed from DNA but not translated into protein(non-coding RNA); instead they are processed from primary transcriptsknown as pri-miRNA to short stem-loop structures called pre-miRNA andfinally to functional miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to downregulate gene expression. MicroRNA sequenceshave been described in publications such as, Lim, et al., Genes &Development, 17, p. 991-1008 (2003), Lim et al Science 299, 1540 (2003),Lee and Ambros Science, 294, 862 (2001), Lau et al., Science 294,858-861 (2001), Lagos-Quintana et al, Current Biology, 12, 735-739(2002), Lagos Quintana et al, Science 294, 853-857 (2001), andLagos-Quintana et al, RNA, 9, 175-179 (2003), which are incorporated byreference. Multiple microRNAs can also be incorporated into theprecursor molecule.

A mature miRNA is produced as a result of a series of miRNA maturationsteps; first a gene encoding the miRNA is transcribed. The gene encodingthe miRNA is typically much longer than the processed mature miRNAmolecule; miRNAs are first transcribed as primary transcripts or“pri-miRNA” with a cap and poly-A tail, which is subsequently processedto short, about 70-nucleotide “stem-loop structures” known as“pre-miRNA” in the cell nucleus. This processing is performed in animalsby a protein complex known as the Microprocessor complex, consisting ofthe nuclease Drosha and the double-stranded RNA binding protein Pasha.These pre-miRNAs are then processed to mature miRNAs in the cytoplasm byinteraction with the endonuclease Dicer, which also initiates theformation of the RNA-induced silencing complex (RISC). This complex isresponsible for the gene silencing observed due to miRNA expression andRNA interference. The pathway is different for miRNAs derived fromintronic stem-loops; these are processed by Drosha but not by Dicer. Insome instances, a given region of DNA and its complementary strand canboth function as templates to give rise to at least two miRNAs. MaturemiRNAs can direct the cleavage of mRNA or they can interfere withtranslation of the mRNA, either of which results in reduced proteinaccumulation, rendering miRNAs capable of modulating gene expression andrelated cellular activities.

The term “pri-miRNA” refers to a precursor to a mature miRNA moleculewhich comprises; (i) a microRNA sequence and (ii) stem-loop componentwhich are both flanked (i.e. surrounded on each side) by “microRNAflanking sequences”, where each flanking sequence typically ends ineither a cap or poly-A tail. A pri-microRNA, (also referred to as largeRNA precursors), are composed of any type of nucleic acid based moleculecapable of accommodating the microRNA flanking sequences and themicroRNA sequence. Examples of pri-miRNAs and the individual componentsof such precursors (flanking sequences and microRNA sequence) areprovided herein. The nucleotide sequence of the pri-miRNA precursor andits stem-loop components can vary widely. In one aspect a pre-miRNAmolecule can be an isolated nucleic acid; including microRNA flankingsequences and comprising a stem-loop structure and a microRNA sequenceincorporated therein. A pri-miRNA molecule can be processed in vivo orin vitro to an intermediate species caller “pre-miRNA”, which is furtherprocessed to produce a mature miRNA.

The term “pre-miRNA” refers to the intermediate miRNA species in theprocessing of a pri-miRNA to mature miRNA, where pri-miRNA is processedto pre-miRNA in the nucleus, where upon pre-miRNA translocates to thecytoplasm where it undergoes additional processing in the cytoplasm toform mature miRNA. Pre-miRNAs are generally about 70 nucleotides long,but can be less than 70 nucleotides or more than 70 nucleotides.

The term “microRNA flanking sequence” as used herein refers tonucleotide sequences including microRNA processing elements. MicroRNAprocessing elements are the minimal nucleic acid sequences whichcontribute to the production of mature miRNA from precursor microRNA.Often these elements are located within a 40 nucleotide sequence thatflanks a microRNA stem-loop structure. In some instances the microRNAprocessing elements are found within a stretch of nucleotide sequencesof between 5 and 4,000 nucleotides in length that flank a microRNAstem-loop structure. Thus, in some embodiments the flanking sequencesare 5-4,000 nucleotides in length. As a result, the length of theprecursor molecule can be, in some instances at least about 150nucleotides or 270 nucleotides in length. The total length of theprecursor molecule, however, can be greater or less than these values.In other embodiments the minimal length of the microRNA flankingsequence is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and anyinteger there between. In other embodiments the maximal length of themicroRNA flanking sequence is 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500,3,600, 3,700, 3,800, 3,900 4,000 and any integer there between.

MicroRNA flanking sequences can be native microRNA flanking sequences orartificial microRNA flanking sequences. A native microRNA flankingsequence is a nucleotide sequence that is ordinarily associated innaturally existing systems with microRNA sequences, i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo. Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking to microRNAsequences in naturally existing systems. microRNA flanking sequenceswithin the pri-miRNA molecule can flank one or both sides of thestem-loop structure encompassing the microRNA sequence. Thus, one end(i.e., 5′) of the stem-loop structure can be adjacent to a singleflanking sequence and the other end (i.e., 3′) of the stem-loopstructure can not be adjacent to a flanking sequence. Preferredstructures have flanking sequences on both ends of the stem-loopstructure. The flanking sequences can be directly adjacent to one orboth ends of the stem-loop structure or can be connected to thestem-loop structure through a linker, additional nucleotides or othermolecules.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (stem portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion). The terms “hairpin” and “fold-back” structures are also usedherein to refer to stem-loop structures. Such structures are well knownin the art and the term is used consistently with its known meaning inthe art. The actual primary sequence of nucleotides within the stem-loopstructure is not critical to the practice of the invention as long asthe secondary structure is present. As is known in the art, thesecondary structure does not require exact base-pairing. Thus, the stemcan include one or more base mismatches. Alternatively, the base-pairingcan be exact, i.e. not include any mismatches. In some instances theprecursor microRNA molecule can include more than one stem-loopstructure. The multiple stem-loop structures can be linked to oneanother through a linker, such as, for example, a nucleic acid linker orby a microRNA flanking sequence or other molecule or some combinationthereof.

Furthermore, miRNA-like stem-loops can be expressed in cells as avehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs)for the purpose of modulating the expression of endogenous genes throughthe miRNA and or RNAi pathways. As used herein, the term “miRNA mimetic”refers to an artificial miRNA or RNAi (RNA interference molecule) whichis flanked by the stem-loop like structures of a pri-miRNA.

The term “artificial microRNA” includes any type of RNA sequence, otherthan endogenous microRNA, which is capable of modulating the productiveutilization of mRNA. For instance, the term artificial microRNA alsoencompasses a nucleic acid sequence which would be previously identifiedas siRNA, where the siRNA is incorporated into a vector and surroundedby miRNA flanking sequences as described herein.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two substantially complementary strands.Double-stranded molecules include those comprised of a single RNAmolecule that doubles back on itself to form a two-stranded structure.For example, the stem loop structure of the progenitor molecules fromwhich the single-stranded miRNA is derived, called the pre-miRNA (Bartelet al. 2004. Cell 116:281-297), comprises a dsRNA molecule.

As used herein, “gene silencing” or “gene silenced” by a miRNA and/orRNA interference molecule “refers to a decrease in the mRNA level in acell for a target gene by at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99% up to andincluding 100%, and any integer in between of the mRNA level found inthe cell without the presence of the miRNA or RNA interference molecule.In one preferred embodiment, the mRNA levels are decreased by at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 99%, up to and including 100% and any integer in between5% and 100%.”

The term “reduced” or “reduce” as used herein generally means a decreaseby a statistically significant amount. However, for avoidance of doubt,“reduced” means a decrease by at least 10% as compared to a referencelevel, for example a decrease by at least about 20%, or at least about30%, or at least about 40%, or at least about 50%, or at least about60%, or at least about 70%, or at least about 80%, or at least about 90%or up to and including a 100% decrease (i.e. absent level as compared toa reference sample), or any decrease between 10-100% as compared to areference level.

The term “lacking” or “lack of” when used in the context of theexpression of let-7 herein, refers to a level let-7 which isundetectable by the methods as used herein to measure such levels. Theterm “lack of” typically refers to minimal, absent or about null levelsof let-7 expression, but does not necessarily mean let-7 is completelyabsent, it means the level of let-7 in a cell is below a level for asignificant let-7 target gene silencing in that cell.

The term “increased” or “increase” as used herein generally means anincrease by a statically significant amount; for the avoidance of anydoubt, “increased” means an increase of at least 10% as compared to areference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

The terms “enriching” or “enriched” are used interchangeably herein andmean that the yield (fraction) of cells of one type is increased by atleast 10% over the fraction of cells of that type in the startingculture or preparation.

The term “tissue” refers to a group or layer of similarly specializedcells which together perform certain special functions. The term“tissue-specific” refers to a source of cells from a specific tissue.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population. Recast, the terms “substantiallypure” or “essentially purified”, with regard to a preparation of one ormore partially and/or terminally differentiated cell types, refer to apopulation of cells that contain fewer than about 20%, more preferablyfewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%,4%, 3%, 2%, 1%, or less than 1%, of cells that are not stem cells orstem cell progeny.

The term “stem cell” as used herein, as used in the context of or withreference to a “cancer stem cell” refers to an undifferentiated cellwhich is capable of proliferation and giving rise to more progenitorcells having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated, or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “cancer stem cell” refers then, to acell with the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell can derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells can be considered stem cells,the range of cell types each can give rise to can vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity can be natural or canbe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stemness.” Self-renewal is the other classical part of thestem cell definition, and it is essential as used in this document. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Formally, it ispossible that cells that begin as stem cells might proceed toward adifferentiated phenotype, but then “reverse” and re-express the stemcell phenotype, a term often referred to as “dedifferentiation”. Cancerstem cells have the ability for self-renewal, multipotentdifferentiation and vigorous proliferative capacity, as well as theability to form mammospheres (or emboid bodies). In some embodiments,breast cancer stem cells are also positive for breast cancer stem cellphenotype (Oct4⁺CD44⁺CD24⁻lineage⁻)¹

The term “progenitor cells” is used synonymously with “stem cell.”Generally, “progenitor cells” have a cellular phenotype that is moreprimitive (i.e., is at an earlier step along a developmental pathway orprogression) than is a fully differentiated cell. Often, progenitorcells also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate. It is possible that cells that begin as progenitorcells might proceed toward a differentiated phenotype, but then“reverse” and re-express the progenitor cell phenotype.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway, and then to an end-stage differentiated cell,which plays a characteristic role in a certain tissue type, and can orcan not retain the capacity to proliferate further.

As indicated above, there are different levels or classes of cellsfalling under the general definition of a “stem cell.” These are“totipotent,” “pluripotent” and “multipotent” stem cells. The term“totipotent” refers to a stem cell that can give rise to any tissue orcell type in the body. “Pluripotent” stem cells can give rise to anytype of cell in the body except germ line cells. Stem cells that cangive rise to a smaller or limited number of different cell types aregenerally termed “multipotent.” Thus, totipotent cells differentiateinto pluripotent cells that can give rise to most, but not all, of thetissues necessary for fetal development. Pluripotent cells undergofurther differentiation into multipotent cells that are committed togive rise to cells that have a particular function. For example,multipotent hematopoietic stem cells give rise to the red blood cells,white blood cells and platelets in the blood.

The term “stemness” as used herein refers to a cell with stem cellproperties, for example a cell that has the capacity for self-renewal,for example a cell that is totipotent, pluripotent or multipotent. Acancer cell that is a “cancer stem cell” or a cancer cell with stemnessproperties is a cancer cell which can give rises to daughter cells whichthemselves can be induced to proliferate and produce progeny thatsubsequently differentiate into one or more mature cell types, whilealso retaining one or more cells with parental developmental potential.The term “cancer stem cell” therefore refers to a cell with the capacityor potential, under particular circumstances, to differentiate to a morespecialized or differentiated phenotype, and which retains the capacity,under certain circumstances, to proliferate without substantiallydifferentiating the self-renewal potential of breast tumor-initiatingcells can be determined by their capacity to give rise to mammospheres¹⁰or emboid bodies in vitro. Furthermore, self-renewing breast cancercells have been shown to be CD44⁺CD24^(−9,10,15), as demonstrated inExample 1 herein. Cancer stem cells have the ability for self-renewal,multipotent differentiation and vigorous proliferative capacity, as wellas the ability to form mammospheres (or emboid bodies).

As used herein a “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a particular gene or target gene when the siRNA isexpressed in the same cell as the gene or target gene. The doublestranded RNA siRNA can be formed by the complementary strands. Thecomplementary portions of the siRNA that hybridize to form the doublestranded molecule typically have substantial or complete identity. Inone embodiment, a siRNA refers to a nucleic acid that has substantial orcomplete identity to sequence of a target gene and forms a doublestranded RNA. The sequence of the siRNA can correspond to the fulllength target gene, or to a subsequence thereof. Typically, the siRNA isat least about 15-50 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is about 15-50 nucleotides inlength, and the double stranded siRNA is about 15-50 base pairs inlength, preferably about 19-30 base nucleotides, preferably about 20-25nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length).

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g. about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

The term “biological sample” as used herein means a sample of biologicaltissue or fluid that comprises nucleic acids. Such samples include, butare not limited to, tissue isolated from animals. Biological samples canalso include sections of tissues such as biopsy and autopsy samples,frozen sections taken for histologic purposes, blood, plasma, serum,sputum, stool, tears, mucus, hair, and skin. Biological samples alsoinclude explants and primary and/or transformed cell cultures derivedfrom patient tissues. A biological sample can be provided by removing asample of cells from an animal, but can also be accomplished by usingpreviously isolated cells (e.g., isolated by another person, at anothertime, and/or for another purpose), or by performing the methods asdisclosed herein in vivo. Archival tissues, such as those havingtreatment or outcome history can also be used.

The term “tissue” is intended to include, blood, blood preparations suchas plasma and serum, bones, joints, muscles, smooth muscles, and organs.

The terms “disease” or “disorder” are used interchangeably herein, andrefer to any alteration in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition oraffliction.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom treatment,including prophylactic treatment, with a pharmaceutical composition asdisclosed herein, is provided. The term “subject” as used herein refersto human and non-human animals. The term “non-human animals” and“non-human mammals” are used interchangeably herein and includes allvertebrates, e.g., mammals, such as non-human primates, (particularlyhigher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig,goat, pig, cat, rabbits, cows, and non-mammals such as chickens,amphibians, reptiles etc. In one embodiment, the subject is human. Inanother embodiment, the subject is an experimental animal or animalsubstitute as a disease model.

The term ‘effective amount” as used herein refers to the amount oftherapeutic agent of pharmaceutical composition to alleviate at leastone of the symptoms of the disease or disorder.

The terms “malignancy” and “cancer” are used interchangeably herein andrefers to any disease of an organ or tissue in mammals characterized bypoorly controlled or uncontrolled multiplication of normal or abnormalcells in that tissue which results in a tumor and has an effect on thebody as a whole. Cancer diseases within the scope of the definitioncomprise benign neoplasms, dysplasias, hyperplasias as well as neoplasmsshowing metastatic growth or any other transformations like e.g.leukoplakias which often precede a breakout of cancer.

As used herein, the term “tumor” refers to a mass of transformed cellsthat are characterized, at least in part, by containing angiogenicvasculature. The transformed cells are characterized by neoplasticuncontrolled cell multiplication which is rapid and continues even afterthe stimuli that initiated the new growth has ceased. The term “tumor”is used broadly to include the tumor parenchymal cells as well as thesupporting stroma, including the angiogenic blood vessels thatinfiltrate the tumor parenchymal cell mass. Although a tumor generallyis a malignant tumor, i.e., a cancer having the ability to metastasize(i.e. a metastatic tumor), a tumor also can be nonmalignant (i.e.non-metastatic tumor). Tumors are hallmarks of cancer, a neoplasticdisease the natural course of which is fatal. Cancer cells exhibit theproperties of invasion and metastasis and are highly anaplastic.

As used herein, the term “metastases” or “metastatic tumor” refers to asecondary tumor that grows separately elsewhere in the body from theprimary tumor and has arisen from detached, transported cells, whereinthe primary tumor is a solid tumor. The primary tumor, as used herein,refers to a tumor that originated in the location or organ in which itis present and did not metastasize to that location from anotherlocation. As used herein, a “malignant tumor” is one having theproperties of invasion and metastasis and showing a high degree ofanaplasia. Anaplasia is the reversion of cells to an immature or a lessdifferentiated form, and it occurs in most malignant tumors.

The term “therapy resistant cancer” as used herein refers to a cancerpresent in a subject which is resistant to, or refractory to at leasttwo different anti-cancer agents such as chemotherapy agents, whichmeans, typically a subject has been treated with at least two differentanti-cancer agents that did not provide effective treatment as that termis defined herein.

The term “gene” as used herein refers to a genomic gene comprisingtranscriptional and/or translational regulatory sequences and/or acoding region and/or non-translated sequences (e.g., introns, 5′- and3′-untranslated sequences). The coding region of a gene can be anucleotide sequence coding for an amino acid sequence or a functionalRNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA.A gene can also be an mRNA or cDNA corresponding to the coding regions(e.g., exons and miRNA) optionally comprising 5′- or 3′ untranslatedsequences linked thereto. A gene can also be an amplified nucleic acidmolecule produced in vitro comprising all or a part of the coding regionand/or 5′- or 3′-untranslated sequences linked thereto.

The term “nucleic acid” or “oligonucleotide” or “polynucleotide” usedherein means at least two nucleotides covalently linked together. Aswill be appreciated by those in the art, the depiction of a singlestrand also defines the sequence of the complementary strand. Thus, anucleic acid also encompasses the complementary strand of a depictedsingle strand. As will also be appreciated by those in the art, manyvariants of a nucleic acid can be used for the same purpose as a givennucleic acid. Thus, a nucleic acid also encompasses substantiallyidentical nucleic acids and complements thereof. As will also beappreciated by those in the art, a single strand provides a probe thatcan hybridize to the target sequence under stringent hybridizationconditions. Thus, a nucleic acid also encompasses a probe thathybridizes under stringent hybridization conditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs can be included that can have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, which are incorporated by reference. Nucleic acids containingone or more non-naturally occurring or modified nucleotides are alsoincluded within one definition of nucleic acids. The modified nucleotideanalog can be located for example at the 5′-end and/or the 3′-end of thenucleic acid molecule. Representative examples of nucleotide analogs canbe selected from sugar- or backbone-modified ribonucleotides. It shouldbe noted, however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e. g. 7 deaza-adenosine; O- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2′OH— group can be replaced by a group selected from H. OR, R. halo, SH,SR, NH2, NHR, NR2 or CN, wherein R is C-C6 alkyl, alkenyl or alkynyl andhalo is F. Cl, Br or I. Modifications of the ribose-phosphate backbonecan be done for a variety of reasons, e.g., to increase the stabilityand half-life of such molecules in physiological environments or asprobes on a biochip. Mixtures of naturally occurring nucleic acids andanalogs can be made; alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogscan be made.

The term “probe” as used herein refers to an oligonucleotide capable ofbinding to a target nucleic acid of complementary sequence through oneor more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. Probes can bind targetsequences lacking complete complementarily with the probe sequencedepending upon the stringency of the hybridization conditions. There canbe any number of base pair mismatches which will interfere withhybridization between the target sequence and single stranded targetnucleic acids, but a probe will bind a selected target specifically,i.e. to the substantial exclusion of non-target nucleic acids under atleast one set of conditions. A probe can be single stranded or partiallysingle and partially double stranded. A probe will generally bedetectably labeled or carry a moiety that permits signal detection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482 (1981), which is incorporated by reference herein), bythe homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-53 (1970), which is incorporated by reference herein), by thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. USA 85:2444-48 (1988), which is incorporated by reference herein),by computerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. (See generally Ausubel et al. (eds.), Current Protocols inMolecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which isincorporated by reference herein). The method used is similar to themethod described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53(1989), which is incorporated by reference herein). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), whichis incorporated by reference herein). (See also Zhang et al., NucleicAcid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res.25:3389-402 (1997), which are incorporated by reference herein).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information internet web site. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.(1990), supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9(1992), which is incorporated by reference herein) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci.USA 90:5873-77 (1993), which is incorporated by reference herein). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance. For example, a nucleic acid is considered similar to areference sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about0.1, more typically less than about 0.01, and most typically less thanabout 0.001.

The term “variant” as used in the context of let-7 miRNA variants meansa modified let-7 miRNA with at least on of the following; alterednucleic acid sequence, such as insertions, deletions, substitutions,fragments of at least 5 nucleic acids, modification of the nucleic acidsor nucleic acid analogues as compared to the wild type mature let-7miRNA (SEQ ID NO:1).

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicates that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment (see below) are identical, with appropriate nucleotideinsertions or deletions or amino-acid insertions or deletions, in atleast 70% of the nucleotides, usually from about 75% to 99%, and morepreferably at least about 98 to 99% of the nucleotides. The term“homolog” or “homologous” as used herein also refers to homology withrespect to structure and/or function. With respect to sequence homology,sequences are homologs if they are at least 50%, at least 60 at least70%, at least 80%, at least 90%, at least 95% identical, at least 97%identical, or at least 99% identical. The term “substantiallyhomologous” refers to sequences that are at least 90%, at least 95%identical, at least 97% identical or at least 99% identical. Homologoussequences can be the same functional gene in different species.

Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan. The terms“homology”, “identity” and “similarity” refer to the degree of sequencesimilarity between two peptides or between two optimally aligned nucleicacid molecules. Homology and identity can each be determined bycomparing a position in each sequence which can be aligned for purposesof comparison. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectfully. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with a sequence of the presentapplication.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G. U. or 1) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “substantial identity” as used herein denotes a characteristicof a polynucleotide or amino acid sequence, wherein the polynucleotideor amino acid comprises a sequence that has at least 85 percent sequenceidentity, preferably at least 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 18 nucleotide (6 aminoacid) positions, frequently over a window of at least 24-48 nucleotide(8-16 amino acid) positions, wherein the; percentage of sequenceidentity is calculated by comparing the reference sequence to thesequence which can include deletions or additions which total 20 percentor less of the reference sequence over the comparison window. Thereference sequence can be a subset of a larger sequence. The term“similarity”, when used to describe a polypeptide, is determined bycomparing the amino acid sequence and the conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

As used herein, the term “expression profile” refers to the amount orlevel of a plurality of different miRNAs expressed in a cell or apopulation of cells or a tissue.

The term “target” as used herein refers to a polynucleotide that can bebound by one or more probes under stringent hybridization conditions.The term “targeting” as used herein in the context of “targeting acancer cell” means directing a therapeutic agent as disclosed herein,such as a let-7 miRNA or homologues thereof to that cancer cell to treata cancer comprising such a cancer stem cell.

The term “target cell” as used herein refers to a cell which comprisescell surface antigens, such as for example but not limited to, cellsurface receptors or glycoprotein or other cell surface markers whichthe targeting moiety as disclosed herein can recognize and bind thereto.

The terms “targeting moiety” or “target moiety” are used interchangeablyherein and refer to a molecule which has affinity, or binds to amolecule on the surface of a target cell, for example a targeting moietyfunctions as an agent that homes in on or preferentially associates orbinds to a particular tissue, cell type, receptor, infecting agent orother area of interest. Examples of a targeting moiety include, but arenot limited to, an antibody, an antigen binding fragment of an antibody,an antigen, a ligand, a receptor, one member of a specific binding pair,a polyamide including a peptide having affinity for a biologicalreceptor, an oligosaccharide, a polysaccharide, a steroid or steroidderivative, a hormone, e.g., estradiol or histamine, a hormone-mimic,e.g., morphine, or other compound having binding specificity for acellular target. In the methods of the present invention, a targetingmoiety promotes transport or preferential localization of the let-7miRNA to a target cell, for example a target cancer stem cell. Targetingmoiety useful in the methods and compositions as disclosed herein bindsto cell-surface antigens or proteins present on cancer stem cells.Examples include, but are not limited to, tumor-associated antigens(TAAs), the HLA-DR antigen, c-erbB-2 proto-oncogene, MUC1, MAG-1,VEGFR2, pro-vasopressin (pro-VP), TAG-72 (sialyl Tn or STn), STn-KLH,GD3, cancer antigen 125 (CA 125, human ovarian cancer cell surfaceantigen. (OCCSA), alpha fetoprotein (AFP), and other cancer cell surfaceantigens which are disclosed in, for example, US20030143237A1, which isincorporated herein by reference.

As used herein, the term “binding moiety” refers to a protein or thenucleic acid binding domain of a protein which has the ability toassociate with or complex with nucleic acids such as let-7 miRNA asdisclosed herein. In some embodiments, a binding moiety is complexedwith a targeting moiety by any means commonly known by persons ofordinary skill in the art, for example but not limited to, fusion,chemical conjugation, van de Waals forces, and in some embodiments thebinding moiety can be fused to the carboxy portion of the targetingmoiety. The location of the targeting moiety may be either in thecarboxyl-terminal or amino-terminal end of the construct or in themiddle of the fusion protein. Alternatively, the fusion protein maycomprise more than one miRNA binding moiety and one or more targetingmoieties. In one embodiment, the binding moiety is the nucleic acidbinding domain of a protein selected from the group of nucleic acidbinding domains present in proteins selected from the group consistingof protamine, GCN4, Fos, Jun, TFIIS, FMRI, yeast protein HX, Vigillin,Mer1, bacterial polynucleotide phosphorylase, ribosomal protein S3, andheat shock protein. In one embodiment, the binding moiety is the proteinprotamine or an nucleic acid-binding fragment of protamine.

The term “vectors” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked; a plasmidis a species of the genus encompassed by “vector”. The term “vector”typically refers to a nucleic acid sequence containing an origin ofreplication and other entities necessary for replication and/ormaintenance in a host cell. Vectors capable of directing the expressionof genes and/or nucleic acid sequence to which they are operativelylinked are referred to herein as “expression vectors”. In general,expression vectors of utility are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome, and typically comprise entities forstable or transient expression or the encoded DNA. Other expressionvectors can be used in the methods as disclosed herein for example, butare not limited to, plasmids, episomes, bacterial artificialchromosomes, yeast artificial chromosomes, bacteriophages or viralvectors, and such vectors can integrate into the host's genome orreplicate autonomously in the particular cell. A vector can be a DNA orRNA vector. Other forms of expression vectors known by those skilled inthe art which serve the equivalent functions can also be used, forexample self replicating extrachromosomal vectors or vectors whichintegrates into a host genome.

As used herein, the terms “treat” or “treatment” or “treating” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow the development of the disease,such as slow down the development of a tumor, the spread of cancer, orreducing at least one effect or symptom of a condition, disease ordisorder associated with inappropriate proliferation or a cell mass, forexample cancer. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced as that term is defined herein.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of at leastslowing of progress or worsening of symptoms that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith cancer, as well as those likely to develop secondary tumors due tometastasis.

The term “effective amount” as used herein refers to the amount oftherapeutic agent of pharmaceutical composition to alleviate at leastone or more symptom of the disease or disorder, and relates to asufficient amount of pharmacological composition to provide the desiredeffect. The phrase “therapeutically effective amount” as used herein,e.g., of an miRNA-related composition as disclosed herein means asufficient amount of the composition to treat a disorder, at areasonable benefit/risk ratio applicable to any medical treatment. Theterm “therapeutically effective amount” therefore refers to an amount ofthe composition as disclosed herein that is sufficient to effect atherapeutically or prophylacticly significant reduction in a symptom orclinical marker associated with a T-cell disease or a cancer-mediatedcondition when administered to a typical subject who has a T-celldisease or a cancer.

A therapeutically or prophylactically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject. Measured or measurableparameters include clinically detectable markers of disease, forexample, elevated or depressed levels of a biological marker, as well asparameters related to a clinically accepted scale of symptoms or markersfor a disease or disorder. It will be understood, however, that thetotal daily usage of the compositions and formulations as disclosedherein will be decided by the attending physician within the scope ofsound medical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated.

With reference to the treatment of a subject with a cancer, the term“therapeutically effective amount” refers to the amount that is safe andsufficient to prevent or delay the development and further growth of atumor or the spread of metastases in cancer patients. The amount canthus cure or cause the cancer to go into remission, slow the course ofcancer progression, slow or inhibit tumor growth, slow or inhibit tumormetastasis, slow or inhibit the establishment of secondary tumors atmetastatic sites, or inhibit the formation of new tumor metastases. Theeffective amount for the treatment of cancer depends on the tumor to betreated, the severity of the tumor, the drug resistance level of thetumor, the species being treated, the age and general condition of thesubject, the mode of administration and so forth. Thus, it is notpossible to specify the exact “effective amount”. However, for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using only routine experimentation. Theefficacy of treatment can be judged by an ordinarily skilledpractitioner, for example, efficacy can be assessed in animal models ofcancer and tumor, for example treatment of a rodent with a cancer, andany treatment or administration of the compositions or formulations thatleads to a decrease of at least one symptom of the cancer, for example areduction in the size of the tumor or a slowing or cessation of the rateof growth of the tumor indicates effective treatment. In embodimentswhere the compositions are used for the treatment of cancer, theefficacy of the composition can be judged using an experimental animalmodel of cancer, e.g., wild-type mice or rats, or preferably,transplantation of tumor cells. When using an experimental animal model,efficacy of treatment is evidenced when a reduction in a symptom of thecancer, for example a reduction in the size of the tumor or a slowing orcessation of the rate of growth of the tumor occurs earlier in treated,versus untreated animals. By “earlier” is meant that a decrease, forexample in the size of the tumor occurs at least 5% earlier, butpreferably more, e.g., one day earlier, two days earlier, 3 daysearlier, or more.

As used herein, the term “treating” when used in reference to a cancertreatment is used to refer to the reduction of a symptom and/or abiochemical marker of cancer, for example a reduction in at least onebiochemical marker of cancer by at least about 10% would be consideredan effective treatment. Examples of such biochemical markers of cancerinclude CD44, telomerase, TGF-α, TGF-β, erbB-2, erbB-3, MUC1, MUC2,CK20, PSA, CA125 and FOBT. A reduction in the rate of proliferation ofthe cancer cells by at least about 10% would also be consideredeffective treatment by the methods as disclosed herein. As alternativeexamples, a reduction in a symptom of cancer, for example, a slowing ofthe rate of growth of the cancer by at least about 10% or a cessation ofthe increase in tumor size, or a reduction in the size of a tumor by atleast about 10% or a reduction in the tumor spread (i.e. tumormetastasis) by at least about 10% would also be considered as affectivetreatments by the methods as disclosed herein. In some embodiments, itis preferred, but not required that the therapeutic agent actually killthe tumor.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of the therapeuticagents as disclosed herein into a subject by a method or route whichresults in delivering of such agent(s) at a desired site. The compoundscan be administered by any appropriate route which results in aneffective treatment in the subject.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration therapeutic compositions other than directly into a tumorsuch that it enters the animal's system and, thus, is subject tometabolism and other like processes.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in maintaining the activity of or carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. In addition to being“pharmaceutically acceptable” as that term is defined herein, eachcarrier must also be “acceptable” in the sense of being compatible withthe other ingredients of the formulation. The pharmaceutical formulationcontains a compound of the invention in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule. Thesepharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration. For the clinical use of themethods of the present invention, targeted delivery composition of theinvention is formulated into pharmaceutical compositions orpharmaceutical formulations for parenteral administration, e.g.,intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical,e.g., transdermal; ocular, e.g., via corneal scarification or other modeof administration. The pharmaceutical composition contains a compound ofthe invention in combination with one or more pharmaceuticallyacceptable ingredients. The carrier can be in the form of a solid,semi-solid or liquid diluent, cream or a capsule.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,ocular parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like. Inaddition, compositions for topical (e.g., oral mucosa, respiratorymucosa) and/or oral administration can form solutions, suspensions,tablets, pills, capsules, sustained-release formulations, oral rinses,or powders, as known in the art are described herein. The compositionsalso can include stabilizers and preservatives. For examples ofcarriers, stabilizers and adjuvants, University of the Sciences inPhiladelphia (2005) Remington: The Science and Practice of Pharmacy withFacts and Comparisons, 21st Ed.

The term “agent” refers to any entity which is normally not present ornot present at the levels being administered to a cell, tissue orsubject. Agent can be selected from a group comprising: chemicals; smallmolecules; nucleic acid sequences; nucleic acid analogues; proteins;peptides; aptamers; antibodies; or functional fragments thereof. Anucleic acid sequence can be RNA or DNA, and can be single or doublestranded, and can be selected from a group comprising: nucleic acidencoding a protein of interest; oligonucleotides; and nucleic acidanalogues; for example peptide-nucleic acid (PNA), pseudo-complementaryPNA (pcPNA), locked nucleic acid (LNA), etc. Such nucleic acid sequencesinclude, but are not limited to nucleic acid sequence encoding proteins,for example that act as transcriptional repressors, antisense molecules,ribozymes, small inhibitory nucleic acid sequences, for example but notlimited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisenseoligonucleotides etc. A protein and/or peptide or fragment thereof canbe any protein of interest, for example, but not limited to; mutatedproteins; therapeutic proteins; truncated proteins, wherein the proteinis normally absent or expressed at lower levels in the cell. Proteinscan also be selected from a group comprising; mutated proteins,genetically engineered proteins, peptides, synthetic peptides,recombinant proteins, chimeric proteins, antibodies, midibodies,tribodies, humanized proteins, humanized antibodies, chimericantibodies, modified proteins and fragments thereof. An gent can beapplied to the media, where it contacts the cell and induces itseffects. Alternatively, an agent can be intracellular as a result ofintroduction of a nucleic acid sequence encoding the agent into the celland its transcription resulting in the production of the nucleic acidand/or protein environmental stimuli within the cell. In someembodiments, the agent is any chemical, entity or moiety, includingwithout limitation synthetic and naturally-occurring non-proteinaceousentities. In certain embodiments the agent is a small molecule having achemical moiety. For example, chemical moieties included unsubstitutedor substituted alkyl, aromatic, or heterocyclyl moieties includingmacrolides, leptomycins and related natural products or analoguesthereof. Agents can be known to have a desired activity and/or property,or can be selected from a library of diverse compounds.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%. The present invention is further explained in detail by thefollowing examples, but the scope of the invention should not be limitedthereto.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Let-7 microRNA

The present invention relates in part to use of let-7 miRNA and/ormimetics thereof for the treatment of cancer. In some embodiments, themethods relate to the treatment of cancer by targeting cancer stem cellswith let-7 miRNA and/or a mimetic thereof. In some embodiments, thelet-7 is any isoform of let-7, including but not limited to let-7a. Inanother embodiment, the let-7 miRNA is a let-7 pre-miRNA, let-7pri-miRNA or mature let-7 miRNA or fragments and variants thereof thatretain the biological activity of the mature let-7 miRNA.

Let-7 microRNAs or let-7 miRNAs are endogenous RNAs which function asgene silencing molecules that regulate the expression of protein-codinggenes that comprise a let-7 target sequence. Let-7 miRNAs function torepress the expression of genes at the posttranscriptional level. Let-7target sites to which let-7 miRNA binds in its role as a gene silencerare typically in the mRNA of the target gene, and can be in the 5′ UTR,the 3′ UTR or in the coding region.

Micro RNAs (also referred to as “miRNAs”) are small non-coding RNAsbelonging to a class of regulatory molecules found in plants andanimals. Without wishing to be bound by theory, miRNAs are thought tocontrol gene expression by binding to complementary sites (hereinreferred to as “target sequences”) on target messenger RNA (mRNA)transcripts. miRNAs often function as “gene silencers” to suppress orrepress the expression of a target gene. miRNAs are generated from largeRNA precursors (termed pri-miRNAs) that are processed in the nucleusinto approximately 70 nucleotide pre-miRNAs, which fold into imperfectstem loop structures (Lee, Y., et al., Nature (2003) 425(6956):415-9)(See FIG. 7 c). The pre-miRNAs undergo an additional processing stepwithin the cytoplasm where mature miRNAs of 18-25 nucleotides in lengthare excised from one side of the pre-miRNA hairpin by an RNase IIIenzyme, Dicer (Hutvagner, G., et al., Science (2001) 12:12 and Grishok,A., et al., Cell (2001) 106(1):23-34).

MiRNAs have been shown to regulate gene expression in two ways. First,miRNAs that bind to protein-coding mRNA sequences that are exactlycomplementary to the miRNA induce the RNA-mediated interference (RNAi)pathway. Messenger RNA targets are cleaved by ribonucleases in the RISCcomplex. This mechanism of miRNA-mediated gene silencing has beenobserved mainly in plants (Hamilton, A. J. and D. C. Baulcombe, Science(1999) 286(5441):950-2 and Reinhart, B. J., et al., MicroRNAs in plants.Genes and Dev. (2002) 16:1616-1626), but an example is known fromanimals (Yekta, S., I. H. Shih, and D. P. Bartel, Science (2004)304(5670):594-6). In the second mechanism, miRNAs that bind to imperfectcomplementary sites on messenger RNA transcripts direct gene regulationat the posttranscriptional level but do not cleave their mRNA targets.MiRNAs identified in both plants and animals use this mechanism to exerttranslational control of their gene targets (Barter, D. P., Cell (2004)116(2):281-97).

Hundreds of miRNAs have been identified in the fly, worm, plant andmammalian genomes. The biological role for the majority of the miRNAsremains unknown because almost all of these were found through cloningand bioinformatic approaches (Lagos-Quintana, M., et al., Curr Biol(2002) 12(9):735-9, Lagos-Quintana, M., et al., RNA (2003) 9(2):175-179, Lagos-Quintana, M., et al., Science (2001) 294(5543): 853-8;Lee, R. C. and V. Ambros, Science (2001) 294(5543):862-4; Lau, N. C., etal., Science (2001) 294(5543):858-62; Lim, L. P., et al., Genes Dev(2003) 17(8):991 1008; Johnston, R. J. and O. Robert, Nature (2003)426(6968):845-9; and Chang, S., et al. Nature (2004) 430(7001):785-9).

let-7 is an endogenous miRNA which functions as a gene silencingmolecule to regulate, at the posttranscriptional level, the expressionof some known protein-coding genes that comprise a let-7 targetsequence. let-7 miRNA and homologues and variants thereof includeconservative substitutions, additions, and deletions therein notadversely affecting the structure or gene silencing function.Preferably, let-7 refers to the nucleic acid encoding let-7 from C.elegans (NCBI Accession No. AY390762), most preferably, let-7 refers tothe nucleic acid encoding a let-7 family member from humans, includingbut not limited to NCBI Accession Nos. AJ421724, AJ421725, AJ421726,AJ421727, AJ421728, AJ421729, AJ421730, AJ421731, AJ421732, andbiologically active sequence variants of let-7, including alleles, andin vitro generated derivatives of let-7 that demonstrate let-7 activityin that it is capable of binding to and inhibiting the expression of agene comprising the let-7 target sequence, where the target sequence is5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID 9). Let-7 also encompasses allisoforms of let-7, for example but not limited to let-7 family membersincluding let-7a (SEQ ID NO:1); let-7b (SEQ ID NO:2); hsa-let-7c (SEQ IDNO:3); hsa-let-7d (SEQ ID NO:4); hsa-let-7e (SEQ ID NO:5); hsa-let-7f(SEQ ID NO:6).

In one embodiment, the let-7 miRNAs useful according to the presentinvention are members of the let-7 family, and can be selected from thegroup consisting of: hsa-let-7a MIMAT0000062:5′-UGAGGUAGUAGGUUGUAUAGUU-3′ (SEQ ID NO: 1); hsa-let-7b MIMAT0000063:5′-UGAGGUAGUAGGUUGUGUGGUU-3′ (SEQ ID NO:2); hsa-let-7c MIMAT0000064:5′-UGAGGUAGUAGGUUGUAUGGUU-3′ SEQ ID NO:3); hsa-let-7d MIMAT0000065:5′-AGAGGUAGUAGGUUGCAUAGU-3′ (SEQ ID NO:4); hsa-let-7e MIMAT0000066:5′-UGAGGUAGGAGGUUGUAUAGU-3′ (SEQ ID NO:5); hsa-let-7f MIMAT0000067:5′-UGAGGUAGUAGAUUGUAUAGUU-3 (SEQ ID NO:6). In some embodiments, thelet-7 miRNA is let-7a isoform, of hsa-let-7a MIMAT0000062:5′-UGAGGUAGUAGGUUGUAUAGUU-3′ (SEQ ID NO:1).

In some embodiments, the let-7 miRNA or let-7 miRNA is a pre-miRNA. Insome embodiments, the let-7 miRNA is5′-UGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUUCCUA-3′ (SEQ ID NO:7), also called MI0000060herein.

In some embodiments, let-7 is homo sapiens let-7a1 stem loop (SEQ IDNO:8) as shown in FIG. 7C, which corresponds to5′-ugggaugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuu-uccua-3′where the underlined residues represent non hybridized nucleic acids,bold resides represent part of the hair-pin turn and the bold underlinedresidues represent the middle residues of hairpin turn (see FIG. 7Cherein).

Let-7 useful in the present invention includes sequence variants oflet-7 that retain at least 50% of the target gene-inhibitory function ofwildtype mature let-7 miRNA (SEQ ID NO: 1). Let-7 variants generallyfall into one or more of three classes: substitution, insertional ordeletional variants. Insertions include 5′ and/or 3′ terminal fusions aswell as intrasequence insertions of single or multiple residues.Insertions can also be introduced within the mature sequence of let-7.Intrasequence insertions ordinarily will be smaller insertions thanthose at the 5′ or 3′ terminus, on the order of 1 to 4 residues. It isunderstood that the variants substitutions insertions or deletions ofresidues will not result in a deleterious effect on the function of thevariant in its ability to bind to, and inhibit the expression of genescomprising let-7 target sequence 5′-AACTATACAACCTACTACCTCA-3′ (SEQ IDNO: 9), and preferably the substitution, insertional or deletionalvariants with have increased binding affinity for the let-7 targetsequence 5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9), and thus increasedgene silencing efficacy of the target gene as compared to wild typelet-7 corresponding to SEQ ID NO:1.

Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Insertionalsequence variants of let-7 are those in which one or more residues areintroduced into a predetermined site in the target let-7 miRNA. Mostcommonly, insertional variants are fusions of nucleic acids at the 5′ or3′ terminus of let-7. Deletion variants are characterized by the removalof one or more residues from the let-7 RNA sequence. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding let-7, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. However,variant let-7 fragments can be conveniently prepared by in vitrosynthesis. The variants typically exhibit the same qualitativebiological activity as the naturally-occurring analogue, althoughvariants also are selected in order to modify the characteristics oflet-7.

While the site for introducing a sequence variation is selected, themutation per se need not be predetermined. For example, in order tooptimize the performance of a mutation at a given site, randommutagenesis can be conducted at the target region and the expressedlet-7 variants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known. Nucleotide substitutions aretypically of single residues; insertions usually will be on the order ofabout from 1 to 10 residues; and deletions will range about from 1 to 30residues. Deletions or insertions preferably are made in adjacent pairs;i.e. a deletion of 2 residues or insertion of 2 residues.

Substitutions, deletion, insertions or any combination thereof can becombined to arrive at a final construct. Changes can be made to increasethe activity of the miRNA, to increase its biological stability orhalf-life, and the like. All such modifications to the nucleotidesequences encoding such miRNA are encompassed.

In some embodiments of the present invention, the let-7 miRNA andmimetics thereof are produced by methods known by persons of ordinaryskill in the art. In some embodiments, the let-7 miRNA and mimeticsthereof can be produced as disclosed in International Patent No:WO2005/047505, which is incorporated herein in its entirety byreference. In some embodiments, the let-7 microRNA molecule is aprecursor microRNA molecule. A let-7 precursor microRNA (let-7pre-miRNA) molecule is an isolated nucleic acid including a stem-loopstructure wherein a microRNA sequence is incorporated into the stem-loopstructure. In some embodiments, the let-7 precursor microRNA moleculeincludes a microRNA flanking sequence on either or both sides of themicroRNA sequence.

In another embodiment, the let-7 microRNA sequence and the microRNAflanking sequence are derived from the same microRNA gene. In anotherembodiment of the invention the let-7 microRNA sequence and the microRNAflanking sequence are not derived from the same microRNA gene.

In another embodiment, a let-7 precursor microRNA has a nucleic acidhaving a stem-loop structure, wherein a let-7 microRNA sequence isincorporated into a stem of the stem-loop structure, and, a microRNAflanking sequence flanking at least one end of the stem-loop structure,wherein the microRNA sequence and the microRNA flanking sequence are notderived from the same microRNA gene.

In some embodiments, the size range of the let-7 miRNA can be from 21nucleotides to 170 nucleotides, although let-7 miRNAs of up to 2000nucleotides can be utilized. In some embodiments the size range of themiRNA is from 70 to 170 nucleotides in length. In another embodiment,mature let-7 miRNAs of from 21 to 25 nucleotides in length can be used.

In some embodiments, the let-7 microRNA sequence is an artificial let-7microRNA. In alternative embodiments, let-7 is from a DNA isolate. A DNAisolate is understood to mean chemically synthesized DNA, cDNA orgenomic DNA with or without the 3′ and/or 5′ flanking regions.

In alternative embodiments, DNA encoding let-7 can be obtained fromother sources by a) obtaining a cDNA library from cells containing mRNA,b) conducting hybridization analysis with labeled DNA encoding let-7 orfragments thereof (usually, greater than 100 bp) in order to detectclones in the cDNA library containing homologous sequences, and c)analyzing the clones by restriction enzyme analysis and nucleic acidsequencing to identify full-length clones.

As used herein nucleic acids and/or nucleic acid sequences arehomologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity is routinely used to establish homology. Higherlevels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or 99% or more can also be used to establish homology. Methods fordetermining sequence similarity percentages (e.g., BLASTN using defaultparameters) are generally available. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov).

Suitable nucleic acids for use in the methods described herein include,but are not limited to, let-7 pri-miRNA, let-7 pre-miRNA, mature let-7miRNA or fragments of variants thereof that retain the biologicalactivity of let-7 miRNA and DNA encoding let-7 pri-miRNA, let-7pre-miRNA, mature let-7 miRNA, fragments or variants thereof, or DNAencoding regulatory elements of let-7 miRNA.

Interestingly, multiple miRNAs can regulate the same mRNA target byrecognizing the same or multiple sites. The presence of multiple miRNAcomplementarily sites in most genetically identified targets canindicate that the cooperative action of multiple RISCs provides the mostefficient translational inhibition. Thus, in one embodiment, the methodsprovide for treatment of cancer by targeting cancer stem cells, themethod comprising targeting the cancer stem cells with a pharmaceuticalcomposition comprising let-7 mimetics, where the let-7 mimetic is adifferent miRNA that complements the function of wild type let-7 miRNA(i.e. SEQ ID NO:1) and/or functions independently of let-7 to genesilence the same mRNA that let-7 silences.

Let-7 Mimetics

In alternative embodiments, mimetics of miRNAs that are reduced and/orlacking in cancer stem cells are useful in the methods of the presentinvention. A miRNA mimetic is any entity or agent that has at least agene-silencing function of the subject miRNA. In some embodiments, let-7mimetics are useful in the methods of the present invention. Examples oflet-7 mimetics include, but are not limited to small molecules,proteins, nucleic acids, ribosomes, aptamers, antibodies, nucleic acidanalogues, etc. that have let-7 activity or function as the term is usedherein. Nucleic acid let-7 mimetics can also include, but are notlimited to, RNA interference-inducing molecules, including, but notlimited to siRNA, dsRNA, stRNA, shRNA and modified versions thereof,where the RNA interference molecule has the same function as let-7miRNA.

In some embodiments the let-7 mimetics can be RNA-interference or RNAinterference molecules, including, but not limited to double-strandedRNA, such as siRNA, double-stranded DNA or single-stranded DNA. In someembodiments, a let-7 mimetic is a single-stranded RNA (ssRNA), a form ofRNA endogenously found in eukaryotic cells as the product of DNAtranscription. Cellular ssRNA molecules include messenger RNAs (and theprogenitor pre-messenger RNAs), small nuclear RNAs, small nucleolarRNAs, transfer RNAs and ribosomal RNAs. Double-stranded RNA (dsRNA)induces a size-dependent immune response such that dsRNA larger than 30bp activates the interferon response, while shorter dsRNAs feed into thecell's endogenous RNA interference machinery downstream of the Dicerenzyme.

Numerous specific siRNA molecules have been designed that have beenshown to inhibit gene expression (Ratcliff et al. Science 276:1558-1560,1997; Waterhouse et al. Nature 411:834-842, 2001). In addition, specificsiRNA molecules have been shown to inhibit, for example, HIV-1 entry toa cell by targeting the host CD4 protein expression in target cellsthereby reducing the entry sites for HIV-1 which targets cellsexpressing CD4 (Novina et al. Nature Medicine, 8:681-686, 2002). Shortinterfering RNA have further been designed and successfully used tosilence expression of Fas to reduce Fas-mediated apoptosis in vivo (Songet al. Nature Medicine 9:347-351, 2003).

It has been shown in plants that longer, about 24-26 nt siRNA,correlates with systemic silencing and methylation of homologous DNA.Conversely, the about 21-22 nt short siRNA class correlates with mRNAdegradation but not with systemic signaling or methylation (Hamilton etal. EMBO J. 2002 Sep. 2; 21(17):4671-9). These findings reveal anunexpected level of complexity in the RNA silencing pathway in plantsthat may also apply in animals. In higher order eukaryotes, DNA ismethylated at cytosines located 5′ to guanosine in the CpG dinucleotide.This modification has important regulatory effects on gene expression,especially when involving CpG-rich areas known as CpG islands, locatedin the promoter regions of many genes. While almost all gene-associatedislands are protected from methylation on autosomal chromosomes,extensive methylation of CpG islands has been associated withtranscriptional inactivation of selected imprinted genes and genes onthe inactive X-chromosomes of females. Aberrant methylation of normallyunmethylated CpG islands has been documented as a relatively frequentevent in immortalized and transformed cells and has been associated withtranscriptional inactivation of defined tumor suppressor genes in humancancers. In this last situation, promoter region hypermethylation standsas an alternative to coding region mutations in eliminating tumorsuppression gene function (Herman, et al.). The use of siRNA moleculesfor directing methylation of a target gene is described in U.S.Provisional Application No. 60/447,013, filed Feb. 13, 2003, referred toin U.S. Patent Application Publication No. 20040091918.

It is also known that the RNA interference does not have to matchperfectly to its target sequence. Preferably, however, the 5′ and middlepart of the antisense (guide) strand of the siRNA is perfectlycomplementary to the target nucleic acid sequence.

The RNA interference-inducing molecule functioning as let-7 mimeticsaccording to the present invention includes RNA molecules that havenatural or modified nucleotides, natural ribose sugars or modifiedsugars and natural or modified phosphate backbone.

Accordingly, the RNA interference-inducing molecules functioning as alet-7 mimetic include, but are not limited to, unmodified and modifieddouble stranded (ds) RNA molecules including short-temporal RNA (stRNA),small interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA(miRNA), and double-stranded RNA (dsRNA), (see, e.g. Baulcombe, Science297:2002-2003, 2002). The dsRNA molecules, e.g. siRNA, also may contain3′ overhangs, preferably 3′UU or 3′TT overhangs. In one embodiment, thesiRNA molecules do not include RNA molecules that comprise ssRNA greaterthan about 30-40 bases, about 40-50 bases, about 50 bases or more. Inone embodiment, the siRNA molecules have a double stranded structure. Inone embodiment, the siRNA molecules are double stranded for more thanabout 25%, more than about 50%, more than about 60%, more than about70%, more than about 80% or more than about 90% of their length.

In some embodiments, let-7 is any agent which binds to and inhibits theexpression of an RNA transcript comprising a let-7 target sequence,where the target sequence is 5′-AACTATACAACCTACTACCTCA-3′ (SEQ ID NO: 9)as shown in FIG. 7D. In such embodiments, these agents can be an RNAinterference-inducing molecule, including, but not limited to unmodifiedand modified double stranded (ds) RNA molecules including,short-temporal RNA (stRNA), small interfering RNA (siRNA), short-hairpinRNA (shRNA), microRNA (miRNA), and double-stranded RNA (dsRNA). In otherembodiments, the agents may be any small molecule, protein, aptamer,nucleic acid analogue, antibody etc. that binds to and inhibits theexpression of an RNA transcript comprising a let-7 target sequence SEQID NO:9.

The miRNA and RNA interference molecules according to the presentinvention can be produced using any known techniques such as directchemical synthesis, through processing of longer double stranded RNAs byexposure to recombinant Dicer protein or Drosophila embryo lysates,through an in vitro system derived from S2 cells, using phage RNApolymerase, RNA-dependant RNA polymerase, and DNA based vectors. Use ofcell lysates or in vitro processing may further involve the subsequentisolation of the short, for example, about 21-23 nucleotide, siRNAs fromthe lysate, etc. Chemical synthesis usually proceeds by making twosingle stranded RNA-oligomers followed by the annealing of the twosingle stranded oligomers into a double stranded RNA. Other examplesinclude methods disclosed in WO 99/32619 and WO 01/68836 that teachchemical and enzymatic synthesis of siRNA. Moreover, numerous commercialservices are available for designing and manufacturing specific siRNAs(see, e.g., QIAGEN Inc., Valencia, Calif. and AMBION Inc., Austin,Tex.).

Examples of methods of preparing such RNA interference are shown, forexample in an International Patent application Nos. PCT/US03/34424 andPCT/US03/34686 the contents and references of which are hereinincorporated by reference in their entirety.

Various specific siRNA and miRNA molecules have been described andadditional molecules can be easily designed by one skilled in the art.For example, the miRNA Database athttp://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml provides auseful source to identify additional miRNAs useful according to thepresent invention (Griffiths-Jones S. NAR, 2004, 32, Database Issue,D109-D111; Ambros V, Bartel B, Bartel D P, Burge C B, Carrington J C,Chen X, Dreyfuss G, Eddy S R, Griffiths-Jones S, Marshall M, Matzke M,Ruvkun G, Tuschl T. RNA, 2003, 9(3), 277-279).

The miRNA and RNA interference as described herein also includes RNAmolecules having one or more non-natural nucleotides, i.e. nucleotidesother than adenine “A”, guanine “G”, uracil “U”, or cytosine “C”, amodified nucleotide residue or a derivative or analog of a naturalnucleotide are also useful. Any modified residue, derivative or analogmay be used to the extent that it does not eliminate or substantiallyreduce (by at least 50%) RNAi activity of the dsRNA. For example, theactivity a miRNA or RNAi molecule with the modified residue can becompared with the activity of a miRNA or RNAi molecule with the samenucleic acid sequence without the modified residue in an assay for genesilencing the target gene. If the miRNA or RNAi with the modifiedresidue(s) has an efficiency of gene silencing which is the same,greater or a least half as efficient as the miRNA or RNAi without themodification, the modified mRNA or RNAi is useful in the methods andcompositions as disclosed herein. Examples of modified residues,derivatives or analogues include, but are not limited to, aminoallylUTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP,alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2′NH2 UTP, 2′NH₂ CTP,and 2′F UTP. Such modified nucleotides include, but are not limited to,aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine,5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine,alpha-S uridine, 4-thio uridine, 2-thio-cytidine, TNH2 uridine, 2′NH₂cytidine, and 2′ F uridine, including the free pho (NTP) RNA moleculesas well as all other useful forms of the nucleotides.

RNA interference as referred to herein additionally includes RNAmolecules which contain modifications in the ribose sugars, as well asmodifications in the “phosphate backbone” of the nucleotide chain. Forexample, siRNA or miRNA molecules containing D-arabinofuranosylstructures in place of the naturally-occurring D-ribonucleosides foundin RNA can be used in RNA interference according to the presentinvention (U.S. Pat. No. 5,177,196). Other examples include RNAmolecules containing the o-linkage between the sugar and theheterocyclic base of the nucleoside, which confers nuclease resistanceand tight complementary strand binding to the oligonucleotides andmolecules similar to the oligonucleotides containing 2′-O-methyl ribose,arabinose and particularly D-arabinose (U.S. Pat. No. 5,177,196). Also,phosphorothioate linkages can be used to stabilize the siRNA and miRNAmolecules (U.S. Pat. No. 5,177,196). siRNA and miRNA molecules havingvarious “tails” covalently attached to either their 3′- or to their5′-ends, or to both, are also been known in the art and can be used tostabilize the siRNA and miRNA molecules delivered using the methods ofthe present invention. Generally speaking, intercalating groups, variouskinds of reporter groups and lipophilic groups attached to the 3′ or 5′ends of the RNA molecules are well known to one skilled in the art andare useful according to the methods of the present invention.Descriptions of syntheses of 3′-cholesterol or 3′-acridine modifiedoligonucleotides applicable to preparation of modified RNA moleculesuseful according to the present invention can be found, for example, inthe articles: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S.,Adams, A. D., Gall, A., Scholler, J. K., and Meyer, R. B. (1993) FacilePreparation and Exonuclease Stability of 3′-ModifiedOligodeoxynucleotides. Nucleic Acids Res. 21 145-150; and Reed, M. W.,Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991) Acridine andCholesterol-Derivatized Solid Supports for Improved Synthesis of3′-Modified Oligonucleotides. Bioconjugate Chem. 2 217-225 (1993).

Let-7 miRNAs and Let-7 Mimetics as Therapeutics

The methods of the invention are useful for treating any type of diseaseor disorder in which it is desirable to increase let-7 miRNA. Theseinclude, for example, diseases where let-7 expression is reduced orlacking in the pathological tissue and the reduction contributes to thedisease pathology and/or progression of the disease. An example of sucha disease is cancer.

In one embodiment methods are provided to treat cancers by using agentswhich inhibit the gene or gene product that is regulated by let-7 miRNA.For example, also encompassed in the present invention is use of anyagent that inhibits the expression of a gene or inhibits its geneproduct (protein) that comprises a let-7 target sequence in its mRNA.The let-7 target site may be in the 5′ UTR, the 3′ UTR or in the codingregion of the mRNA. The target sequence of let-7 is SEQ ID NO:9 orhomologues thereof. Examples of genes that comprise let-7 targetsequences include, but are not limited to, RAS, HRAS, lin-42, KRAS,GRB2, hbl-1, daf-12, pha-4, or human homologues thereof, as disclosed inInternational Patent Application: WO06/028967, which is incorporated inits entirety herein by reference. In some embodiments, these genesencode endogenous mammalian proteins, C. elegans proteins, parasiticproteins, and viral proteins encoded by a eukaryotic cell after entry ofa virus into the cell.

In some embodiments, the subject can be administered a plurality ofagents to inhibit more than one gene and/or protein which are normallyregulated at the level of mRNA by let-7 miRNA. The agents can be RNAinterference molecules, for example miRNA, siRNA, shRNA, or proteins,small molecules, nucleic acids, nucleic acid analogues, aptamers,antibodies, peptides and variants and analogues thereof. In someembodiments, where the agent is an antibody, the antibody can be arecombinant antibody, humanized antibody, chimeric antibody, modifiedantibody, monoclonal antibody, polyclonal antibody, miniantibody,dimeric miniantibody, minibody, diabody or tribody or antigen-bindingvariants, analogues or modified versions thereof.

In some embodiments, the disease is associated with a stem cell. In someembodiments, the disease is associated with a cancer stem cell.

In one embodiment, the cancer is breast cancer. Thus in one embodiment,let-7 miRNA and let-7 mimetics of the present invention are useful intherapeutic protocols in the treatment of breast cancer. In someembodiments, the methods of the present invention are useful fortreating cancers where cancer cells lack let-7 or have reduced let-7expression, including, as non-limiting examples, colon and lung cancer.Thus in one embodiment, let-7 miRNA and let-7 mimetics of the presentinvention are useful in therapeutic protocols in the treatment of, e.g.,colon and lung cancer.

The let-7 miRNA relationship to cancer is not limited to breast, lung,or colon cancer—rather, let-7 miRNA represents a broad-spectrum tumorsuppressor. Thus, in other embodiments, the let-7 miRNA and let-7mimetics of the present invention are useful in therapeutic protocolsrelated to other cancers, including, but not limited to, cancer selectedbreast cancer, lung cancer, head and neck cancer, bladder cancer,stomach cancer, cancer of the nervous system, bone cancer, bone marrowcancer, brain cancer, colon cancer, esophageal cancer, endometrialcancer, gastrointestinal cancer, genital-urinary cancer, stomach cancer,lymphomas, melanoma, glioma, bladder cancer, pancreatic cancer, gumcancer, kidney cancer, retinal cancer, liver cancer, nasopharynx cancer,ovarian cancer, oral cancers, bladder cancer, hematological neoplasms,follicular lymphoma, cervical cancer, multiple myeloma, osteosarcomas,thyroid cancer, prostate cancer, colon cancer, prostate cancer, skincancer, stomach cancer, testis cancer, tongue cancer, or uterine cancer.

Also encompassed is the use of other miRNAs and mimetics thereof thatare downregulated in cancer stem cells for the treatment of othercancers, wherein the cancer comprises a cancer stem cell lacking orhaving reduced expression of the miRNA. Examples of such miRNAs include,but are not limited to miR-107, miR-10a, miR-128a, miR128b, miR-132,miR-138, miR-16, miR-17, miR-195, miR-199a, miR-20, miR-200a, miR-200b,miR-200c, miR-20b, and miR-22.

Production of Let-7 miRNA and Mimetics Thereof

MiRNA can be isolated from cells or tissues, recombinantly produced, orsynthesized in vitro by a variety of techniques well known to one ofordinary skill in the art. In one approach, miRNA is isolated from cellsor tissues.

Techniques for isolating miRNA from cells or tissues are well known toone of ordinary skill in the art. For example, miRNA can be isolatedfrom total RNA using the miRNA isolation kit from Ambion, Inc. Anothertechnique utilizes the flashPAGE Fractionator System (Ambion, Inc.) forPAGE purification of small nucleic acids.

The miRNA can be obtained by preparing a recombinant version thereof(i.e., by using the techniques of genetic engineering to produce arecombinant nucleic acid which can then be isolated or purified bytechniques well known to one of ordinary skill in the art). Thisapproach involves growing a culture of host cells in a suitable culturemedium, and purifying the miRNA from the cells or the culture in whichthe cells are grown. For example, the methods include a process forproducing a miRNA in which a host cell, containing a suitable expressionvector that includes a nucleic acid encoding an miRNA, is cultured underconditions that allow expression of the encoded miRNA. In a preferredembodiment the nucleic acid encodes let-7. The miRNA can be recoveredfrom the culture, from the culture medium or from a lysate prepared fromthe host cells, and further purified. The host cell can be a highereukaryotic host cell such as a mammalian cell, a lower eukaryotic hostcell such as a yeast cell, or the host cell can be a prokaryotic cellsuch as a bacterial cell. Introduction of a vector containing thenucleic acid encoding the miRNA into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)).

Any host/vector system can be used to express one or more of the miRNAs.These include, but are not limited to, eukaryotic hosts such as HeLacells and yeast, as well as prokaryotic host such as E. coli and B.subtilis. miRNA can be expressed in mammalian cells, yeast, bacteria, orother cells where the miRNA gene is under the control of an appropriatepromoter. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989). In the preferred embodiment, the miRNA is expressedin mammalian cells. Examples of mammalian expression systems includeC127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney293 cells, human epidermal A43 1 cells, human Colo205 cells, 3T3 cells,CV-1 cells, other transformed primate cell lines, normal diploid cells,cell strains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BILK, HL-60, U937, HaK or Jurkatcells.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter, polyadenylation site, transcriptional terminationsequences, and 5′ flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example, SV40 origin, earlypromoter, enhancer, splice, and polyadenylation sites may be used toprovide the required non-transcribed genetic elements. Potentiallysuitable yeast strains include Saccharomyces cerevsiae,Schizosaccharomyces pombe, Klayveromyces strains, Candida, or any yeaststrain capable of expressing miRNA. Potentially suitable bacterialstrains include Escherichia coli, Bacillus subtilis, Salmonellatyphimurium, or any bacterial strain capable of expressing miRNA.

In another approach, genomic DNA encoding let-7 is isolated, the genomicDNA is expressed in a mammalian expression system, and RNA is purifiedand modified as necessary for administration to a patient. In oneapproach, the let-7 is in the form of a pre-miRNA, which can be modifiedas desired (i.e. for increased stability or cellular uptake).

Knowledge of DNA sequences of miRNA allows for modification of cells topermit or increase expression of an endogenous miRNA. Cells can bemodified (e.g., by homologous recombination) to provide increased miRNAexpression by replacing, in whole or in part, the naturally occurringpromoter with all or part of a heterologous promoter so that the cellsexpress the miRNA at higher levels. The heterologous promoter isinserted in such a manner that it is operatively linked to the desiredmiRNA encoding sequences. See, for example, PCT InternationalPublication No. WO 94/12650 by Transkaryotic Therapies, Inc., PCTInternational Publication No. WO 92/20808 by Cell Genesys, Inc., and PCTInternational Publication No. WO 91/09955 by Applied Research Systems.Cells also may be; engineered to express an endogenous gene comprisingthe miRNA under the control of inducible regulatory elements, in whichcase the regulatory sequences of the endogenous gene may be replaced byhomologous recombination. Gene activation techniques are described inU.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwinet al.; PCT/US92/09627 (WO93/09222) by Selden et al.; and PCT/US90/06436(WO91/06667) by Skoultchi et al. The miRNA may be prepared by culturingtransformed host cells under culture conditions suitable to express themiRNA. The resulting expressed miRNA may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the miRNA may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; immunoaffnitychromatography, or complementary cDNA affinity chromatography.

The miRNA can also be expressed as a product of transgenic animals,which are characterized by somatic or germ cells containing a nucleotidesequence encoding the miRNA. A vector containing DNA encoding miRNA andappropriate regulatory elements can be inserted in the germ line ofanimals using homologous recombination (Capecchi, Science t244:1288-1292 (1989)), such that they express the miRNA. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 to Robinson, et al., and PCTPublication No. WO 94/28122 by Ontario Cancer Institute. miRNA can beisolated from cells or tissue isolated from transgenic animals asdiscussed above.

In one approach, the miRNA can be obtained synthetically, for example,by chemically synthesizing a nucleic acid by any method of synthesisknown to the skilled artisan. The synthesized miRNA can then be purifiedby any method known in the art. Methods for chemical synthesis ofnucleic acids include, but are not limited to, in vitro chemicalsynthesis using phosphotriester, phosphate or phosphoramidite chemistryand solid phase techniques, or via deoxynucleoside H-phosphonateintermediates (see U.S. Pat. No. 5,705,629 to Bhongle).

In some circumstances, for example, where increased nuclease stabilityis desired, nucleic acids having nucleic acid analogs and/or modifiedinternucleoside linkages may be preferred. Nucleic acids containingmodified internucleoside linkages can also be synthesized using reagentsand methods that are well known in the art. For example, methods ofsynthesizing nucleic acids containing phosphonate phosphorothioate,phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate,formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate,dimethylene-sulfide (—CH2-S—CH2), diinethylene-sulfoxide (—CH2-SO—CH2),dimethylene-sulfone (—CH2-SO2-CH2), 2′-O-alkyl, and 2′-deoxy-2′-fluoro′phosphorothioate internucleoside linkages are well known in the art (seeUhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990,Tetrahedron Lett. 31:335 and references cited therein). U.S. Pat. Nos.5,614,617 and 5,223,618 to Cook, et al., U.S. Pat. No. 5,714,606 toAcevedo, et al, U.S. Pat. No. 5,378,825 to Cook, et al., U.S. Pat. Nos.5,672,697 and 5,466,786 to Buhr, et al., U.S. Pat. No. 5,777,092 toCook, et al., U.S. Pat. No. 5,602,240 to De Mesmacker, et al., U.S. Pat.No. 5,610,289 to Cook, et al. and U.S. Pat. No. 5,858,988 to Wang, alsodescribe nucleic acid analogs for enhanced nuclease stability andcellular uptake.

Pharmaceutical Compositions

In some embodiments, miRNAs and miRNA mimetics, for example let-7 miRNAsand/or let-7 mimetics are administered to a subject in a pharmaceuticalcomposition where the subject has a cancer stem cell. The administrationmay be a treatment and/or prophylaxis for cancer, where the subject hasat least one cancer stem cell. The subject may have, or may not have,symptoms or manifestation of cancer, since cancer stem cells can existin the absence of symptoms of cancer. In some embodiments, thepharmaceutical compositions comprising miRNA and miRNA mimetics, forexample let-7 miRNA and/or let-7 mimetics are administered to a subjectwith a cancer stem cell. In some embodiments, the cancer stem cell ispresent in any type of cancer. In some embodiments, the cancer is breastcancer. In some embodiments, the cancer is a treatment-resistant cancer,for example but not limited to a chemotherapy-resistant breast cancer.

In another aspect of the present invention, the methods described hereinencompass administering a pharmaceutical composition comprising a miRNAor mimetic thereof to a subject, where the subject comprises a cancerstem cell which is lacking or has reduced expression of an miRNA. Inthis aspect, the cancer stem cell is, e.g., a cancer stem cell lackingor having reduced expression of one or more of the following miRNAs;let-7, miR-107, miR-10a, miR-128a, miR128b, miR-132, miR-138, miR-16,miR-17, miR-195, miR-199a, miR-20, miR-200a, miR-200b, miR-200c,miR-20b, miR-22.

Therefore in some embodiments, the methods of the present inventionrelate to the treatment of cancers by targeting cancer stem cells, themethod comprising upregulating let-7 microRNAs or providing analogouspharmaceutical compounds, for example pharmaceutical compositionscomprising let-7 miRNA and/or let-7 mimetics to a cancer stem cell in atherapeutic effective amount for the treatment of cancer. In suchembodiments, the cancer comprises a cancer stem cell and/or a cell withreduced or lacking let-7 expression.

In some embodiments, the let-7 miRNAs are nucleic acids, for example butnot limited to let-7 pri miRNA, let-7 pre-miRNA, mature let-7 miRNA orfragments or variants thereof that retain the same biological activityas the mature let-7 miRNA, for example, they have a minimum biologicalactivity of binding to (or hybridizing to) a target RNA transcriptcomprising a let-7 target sequence, and inhibiting expression from thattranscript where the target sequence is 5′-AACTATACAACCTACTACCTCA-3′(SEQ ID NO: 9). In some embodiments, the let-7 miRNA are encoded by DNAcompositions encoding a let-7 pri miRNA, let-7 pre-miRNA, mature let-7miRNA or fragments or homologues thereof, or regulatory elements whichexpress the let-7 miRNA.

In alternative embodiments, mimetics of let-7 miRNA are useful in themethods of the present invention. A let-7 mimetic is any entity or agentthat functions as a let-7 miRNA. Examples of let-7 mimetics are, but notlimited to small molecules, proteins, nucleic acids, ribosomes,aptamers, antibodies, nucleic acid analogues etc. Nucleic acids let-7mimetics can also be, for example, but not limited to, RNAinterference-inducing molecules, for example but not limited to siRNA,dsRNA, stRNA, shRNA and modified versions thereof, where the RNAinterference molecule has the same function as let-7 miRNA.

In another aspect of the present invention provide methods to treatcancers by targeting the cancer stem cells, the method comprisingtargeting the cancer stem cell with agents that inhibit genes and/ortheir gene products (mRNAs or proteins) which are normally gene silencedby let-7 miRNA. Such genes are, for example, genes which comprise alet-7 target sequence within their mRNA. The let-7 target sequence canbe in the 5′UTR, 3′UTR or coding sequence. Examples of such genes thatcomprise a let-7 target sequence, (i.e. genes which have SEQ ID NO:9 ora homologue thereof) within their mRNA are for example, but not limitedto, RAS, HRAS, KRAS, lin-42, GRB2, hbl-1, daf-12 and pha-4. In someembodiments, agents inhibit the activity and/or the expression of genescomprising let-7 target sequence within their mRNA. In some embodiments,the methods of the present intervention relate to the treatment ofcancers by targeting cancer stem cells, the method comprisingadministering a pharmaceutical composition comprising at least one agentthat inhibits the activity and/or the expression of at least one genethat is gene silenced by let-7 and/or comprises a let-7 target withintheir mRNA to a cancer stem cell in therapeutic effective amount for thetreatment of cancer. In such embodiments, the cancer comprises a cancerstem cell and/or a cell with reduced or lacking let-7 expression.

Effective, safe dosages can be experimentally determined in modelorganisms and in human trials by methods well known to one of ordinaryskill in the art. The let-7 miRNA and let-7 mimetics in a pharmaceuticalcomposition can be administered alone or in combination with adjuvantcancer therapy such as surgery, chemotherapy, radiotherapy,thermotherapy, immunotherapy, hormone therapy and laser therapy, toprovide a beneficial effect, e.g. reduce tumor size, reduce cellproliferation of the tumor, inhibit angiogenesis, inhibit metastasis, orotherwise improve at least one symptom or manifestation of the disease.

In some embodiments, the pharmaceutical compositions comprising let-7miRNA and/or let-7 are administered to cells in vivo and in vitro. Thein vivo administration as used herein means delivery of the let-7 miRNAand let-7 mimetics into a living subject, including human. The in vitroadministration as used herein means delivery of let-7 miRNA and let-7mimetics into cells and organs outside a living subject.

Targeting Let-7 miRNA and Let-7 Mimetics

In another embodiment of the invention miRNA and/or miRNA mimetics, forexample let-7 miRNA and/or let-7 mimetics are targeted to specificcells, for example cancer stem cells in order minimize or avoid anyundesired potential side effects of let miRNA and/or let-7 mimetic. Insuch embodiments, the methods of the present invention provide means totarget cancer stem cells specifically, because these cancer stem cellstypically express a variety of specific proteins on their surface andthus can be targeted. In some embodiments, the miRNA and/or let-7mimetics can be fused to a cell targeting moiety or protein, asdisclosed in the International Patent Application PCT/US05/029111 whichis incorporated herein in its entirety by reference.

In such embodiments, the target moiety specifically brings the deliverysystem to the target cell. The particular target moiety for deliveringthe interference RNAs, including let-7 miRNAs and let-7 mimetic, can bedetermined empirically based upon the present disclosure and dependingupon the target cell. For example, with somatic cell therapy in vivowith readily accessible cells or tissues such as an intravasculartarget, immune cell target or the like, the important attributes of thetarget moiety are affinity and selectivity.

In some embodiments of the present invention, the miRNA and siRNA aredelivered to a limited number of cells thereby limiting, for example,potential side effects of therapies using siRNA. The particular cellsurface targets that are chosen for the targeting moiety will dependupon the target cell. Cells can be specifically targeted, for example,by use of antibodies against unique proteins, lipids or carbohydratesthat are present on the cell surface. A skilled artisan can readilydetermine such molecules based on the general knowledge in the art.

The strategy for choosing the targeting moiety is very adaptable. Forexample, any cell-specific antigen, including proteins, carbohydratesand lipids can be used to create an antibody that can be used to targetthe miRNA and siRNA to a specific cell type according to the methodsdescribed herein. For example, certain tumors frequently possess a largeamount of a particular cell surface receptor (e.g. neu with breastcancers), or an abnormal form of a particular protein. Therefore, atumor antigen can serve as a specific target to deliver siRNA into thetumor cells to inhibit growth and/or proliferation of the cell or todestroy the cell. Any known tumor antigen expressed on the tumor cellsurface can be used for generating an antibody to serve as a targetingmoiety.

For example, tumor antigens useful according to the present inventioninclude, but are not limited to, mini-MUC; MUC-1 (Marshall et al., J.CLin. Oncol. 18:3964-73 (2000); HER2/neu; HER2 receptor (U.S. Pat. No.5,772,997); mammoglobulin (U.S. Pat. No. 5,922,836); labyrinthin (U.S.Pat. No. 6,166,176); SCP-1 (U.S. Pat. No. 6,140,050); NY-ESO-1 (U.S.Pat. No. 6,140,050); SSX-2 (U.S. Pat. No. 6,140,050); N-terminal blockedsoluble cytokeratin (U.S. Pat. No. 4,775,620); 43 kD human cancerantigen (U.S. Pat. No. 6,077,950); human tumor associated antigen (PRAT)(U.S. Pat. No. 6,020,478); human tumor associated antigen (TUAN) (U.S.Pat. No. 5,922,566); L6 antigen (U.S. Pat. No. 5,597,707);carcinoembryonic antigen (RT-PCR analysis for breast cancer prognosis inClin Cancer Res 6:4176-85, 2000); CA15-3 (Eur J Gynaecol Oncol21:278-81, 2000); oncoprotein 18/stathmin (Op18) (Br J. Cancer 83:311-8,2000); human glandular kallikrein (hK2) (Breast Cancer Res Treat59:263-70, 2000); NY-BR antigens (Cancer Immun. Mar. 30; 1:4, 2001),tumor protein D52 (Cancer Immun. March 30; 1:4, 2001), andprostate-specific antigen (Breast Cancer Res Treat 59:263-70, 2000); andEEA.

In some embodiments, the tumor antigens useful for targeting the let-7miRNA and mimetics thereof are CD44, CD133, ABC7, c-kit, or SCA1.

In other embodiments, the let-7 miRNA and/or let-7 mimetics of thepresent invention can be targeted to other receptors of interest, forexample but not limited to include those for lymphokines such asinterleukins and interferons, for example, the interleukin-2 (IL-2)receptor (IL-2R). The p55, IL-2R alpha chain, also referred to as theTac protein, is associated with Ag or mitogen-activated T-cells but notresting T-cells. It is expressed in high levels on malignant cells oflymphoid cancers such as adult T-cell leukemia, cutaneous T-celllymphoma and Hodgkin's disease. The anti-Tac antibody will bind to thisprotein. Humanized version of such antibodies are known and described inQueen, C., et al., Proc. Natl. Acad. Sci. USA:10029-10039 (1989);Hakimi, J., et al., J. of Immun. 151:1075-1085 (1993) (Mik.beta.1 whichis a Mab against IL-2R .beta. chain); Kreitman, R. J., et al., J. ofImmun. 149:2810-2815 (1992); Hakimi, J., et al., J. of Immun.147:1352-1359 (1991). Antibodies to these various proteins are known andavailable. These antibodies can readily be adapted for use in thissystem by following the general procedures described herein, andsubstituting the gene coding for the desired binding site for theexemplified gene.

In another embodiment, let-7 miRNA and/or let-7 mimetics of the presentinvention can be targeted the using single chain antibody fragment, ML39scFv, that recognizes the ErbB2 receptor (Li et al. “Single-chainantibody-mediated gene delivery into ErbB2-positive human breast cancercells” Cancer Gene Ther. 2001; 8:555-65; also Song Nature Biotech 2005).ML39 scFV recognizes the ErbB2 receptor and as such is useful as atargeting moiety in the methods of the present invention for targetingand delivery to cells expressing ErbB2, for example, breast cancercells. Methods for producing a fusion protein containing an ML39 scFvtargeting moiety are described below and in Li et al. 2001 (supra).Other useful single chain antibody fragment to target the let-7 miRNAand RNA interference molecules of the present invention are a singlechain antibody fragment to the transferrin receptor described in, forexample, Xu et al. (Mol Cancer Ther. 2002, 1(5):337-46) and the singlechain antibody fragment recognizing prostate specific membrane antigendescribed in, for example, Li et al. (Intl J Oncology. 2003, 23:1329-1332). Any antibody with a known sequence can be used to prepare asimilar construct as described above, and any method to prepare such aconstruct is commonly known in the art.

Delivery of Let-7 miRNA or Let-7 Mimetics

In one embodiment, a vector encoding let-7 miRNA and/or a let-7 mimeticis delivered into a specific target cell. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), often along with other sequences. Eukaryotic cellsare known to utilize promoters, enhancers, and termination andpolyadenylation signals.

One can also use localization sequences to deliver the released let-7miRNA and/or let-7 mimetics intracellularly to a cell compartment ofinterest. Typically, the delivery system first binds to a specificreceptor on the cell. Thereafter, the targeted cell internalizes thedelivery system, which is bound to the cell. For example, membraneproteins on the cell surface, including receptors and antigens can beinternalized by receptor mediated endocytosis after interaction with theligand to the receptor or antibodies. (Dautry-Varsat, A., et al., Sci.Am. 250:52-58 (1984)). This endocytic process is exploited by thepresent delivery system. Because this process may damage the let-7 miRNAor let-7 RNA interference molecules, for example let-7 siRNA as it isbeing internalized, it may be desirable to use a segment containingmultiple repeats of the RNA interference-inducing molecule of interest.One can also include sequences or moieties that disrupt endosomes andlysosomes. See, e.g., Cristiano, R. J., et al., Proc. Natl. Acad. Sci.USA 90:11548-11552 (1993); Wagner, E., et al., Proc. Natl. Acad. Sci.USA 89:6099-6103 (1992); Cotten, M., et al., Proc. Natl. Acad. Sci. USA89:6094-6098 (1992).

In some embodiments, let-7 miRNA and/or let-7 mimetics are complexedwith desired targeting moieties by mixing the let-7 miRNA or let-7 RNAinterference molecules with the targeting moiety in the presence ofcomplexing agents. Examples of such complexing agents include, but arenot limited to, poly-amino acids; polyimines; polyacrylates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses and starches. In some embodiments, the complexingagents include chitosan, N-trimethylchitosan, poly-L-lysine,polyhistidine, polyornithine, polyspermines, protamine,polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE),polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),poly(ethylcyanoacrylate), poly(butylcyanoacrylate),poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin andDEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lacticacid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, andpolyethyleneglycol (PEG), and polyethylenimine.

In alternative embodiments, let-7 miRNA and/or let-7 mimetic complexingagent is protamine or an RNA-binding domain, such as an siRNA-bindingfragment or nucleic acid binding fragment of protamine. Protamine is apolycationic peptide with molecular weight about 4000-4500 Da. Protamineis a small basic nucleic acid binding protein, which serves to condensethe animal's genomic DNA for packaging into the restrictive volume of asperm head (Warrant, R. W., et al., Nature 271:130-135 (1978); Krawetz,S. A., et al., Genomics 5:639-645 (1989)). The positive charges of theprotamine can strongly interact with negative charges of the phosphatebackbone of nucleic acid, such as RNA, resulting in a neutral and stableinterference RNA-protamine complex.

In one embodiment, the protamine fragment is encoded by a nucleic acidsequence disclosed in International Patent Application: PCT/US05/029111,which is incorporated herein in its entirety by reference. The methods,reagents and references that describe a preparation of a nucleicacid-protamine complex in detail are disclosed in the U.S. PatentApplication Publication Nos. US2002/0132990 and US2004/0023902, and areherein incorporated by reference in their entirety.

In some embodiments, a binding domain is used to complex the targetingmoiety to the let-7 miRNA and/or let-7 mimetic. In some embodiments, thebinding domain is selected from the nucleic acid binding domains presentin proteins selected from the group consisting of GCN4, Fos, Jun, TFIIS,FMRI, yeast protein HX, Vigillin, Mer1, bacterial polynucleotidephosphorylase, ribosomal protein S3, and heat shock protein.

Administration

In one aspect, the invention provides methods of administering any ofthe let-7 miRNA and/or let-7 mimetics described herein to a subject.When administered, the let-7 miRNA and/or let-7 mimetics are applied ina therapeutically effective, pharmaceutically acceptable amount as apharmaceutically acceptable formulation.

A therapeutically effective amount can be determined on an individualbasis and will be based, at least in part, on consideration of thespecies of mammal, the mammal's age, sex, size, and health; the compoundand/or composition used, the type of delivery system used; the time ofadministration relative to the severity of the disease; and whether asingle, multiple, or controlled-release dose regimen is employed. Atherapeutically effective amount can be determined by one of ordinaryskill in the art employing such factors and using no more than routineexperimentation.

In administering the systems and methods of the invention to a subject,dosing amounts, dosing schedules, routes of administration, and the likemay be selected so as to affect known activities of these systems andmethods. Dosage may be adjusted appropriately to achieve desired druglevels, local or systemic, depending upon the mode of administration.The doses may be given in one or several administrations per day. As oneexample, if daily doses are required, daily doses may be from about 0.01mg/kg/day to about 1000 mg/kg/day, and in some embodiments, from about0.1 to about 100 mg/kg/day or from about 1 mg/kg/day to about 10mg/kg/day. Parenteral administration, in some cases, may be from one toseveral orders of magnitude lower dose per day, as compared to oraldoses. For example, the dosage of an active compound when parenterallyadministered may be between about 0.1 micrograms/kg/day to about 10mg/kg/day, and in some embodiments, from about 1 microgram/kg/day toabout 1 mg/kg/day or from about 0.01 mg/kg/day to about 0.1 mg/kg/day.In some embodiments, the concentration of the active compound(s), ifadministered systemically, is at a dose of about 1.0 mg to about 2000 mgfor an adult of kg body weight, per day. In other embodiments, the doseis about 10 mg to about 1000 mg/70 kg/day. In yet other embodiments, thedose is about 100 mg to about 500 mg/70 kg/day. Preferably, theconcentration, if applied topically, is about 0.1 mg to about 500 mg/gmof ointment or other base, more preferably about 1.0 mg to about 100mg/gm of base, and most preferably, about 30 mg to about 70 mg/gm ofbase. The specific concentration partially depends upon the particularcomposition used, as some are more effective than others. The dosageconcentration of the composition actually administered is dependent atleast in part upon the particular physiological response being treated,the final concentration of composition that is desired at the site ofaction, the method of administration, the efficacy of the particularcomposition, the longevity of the particular composition, and the timingof administration relative to the severity of the disease. Preferably,the dosage form is such that it does not substantially deleteriouslyaffect the mammal. The dosage can be determined by one of ordinary skillin the art employing such factors and using no more than routineexperimentation. In the event that the response of a particular subjectis insufficient at such doses, even higher doses (or effectively higherdoses by a different, more localized delivery route) may be employed tothe extent that subject tolerance permits. Multiple doses per day arealso contemplated in some cases to achieve appropriate systemic levelswithin the subject or within the active site of the subject. In somecases, dosing amounts, dosing schedules, routes of administration, andthe like may be selected as described herein, whereby therapeuticallyeffective levels for the treatment of cancer are provided.

In certain embodiments where cancers are being treated, let-7 miRNAand/or let-7 mimetics of the invention may be administered to a subjectwho has a family history of cancer, or to a subject who has a geneticpredisposition for cancer, for example breast cancer. In otherembodiments, let-7 miRNA and/or let-7 mimetics are administered to asubject who has reached a particular age, or to a subject more likely toget cancer. In yet other embodiments, the let-7 miRNA and/or let-7mimetics are administered to subjects who exhibit symptoms of cancer(e.g., early or advanced). In still other embodiments, the compositionmay be administered to a subject as a preventive measure.

In some embodiments, the inventive composition may be administered to asubject based on demographics or epidemiological studies, or to asubject in a particular field or career. In some embodiments, the miRNAand/or miRNA mimetic, for example let-7 or let-7 mimetic areadministered to a subject that has had a prior therapy, for examplecancer therapy. Examples of such therapies include, but are not limitedto, surgery, chemotherapy, radiotherapy, thermotherapy, immunotherapy,hormone therapy and laser therapy.

Administration of let-7 miRNA and/or let-7 mimetics of the invention toa subject may be accomplished by any medically acceptable method whichallows the composition to reach its target. The particular mode selectedwill depend of course, upon factors such as those previously described,for example, the particular composition, the severity of the state ofthe subject being treated, the dosage required for therapeutic efficacy,etc. As used herein, a “medically acceptable” mode of treatment is amode able to produce effective levels of the active compound(s) of thecomposition within the subject without causing clinically unacceptableadverse effects.

The methods to deliver let-7 miRNA and mimetics thereof to the cell orsubject useful in the present invention are well known in the art, andinclude chemical transfection using lipid-based, amine based and polymerbased techniques, viral vectors and combinations thereof (see, forexample, products from Ambion Inc., Austin, Tex.; and Novagen, EMDBiosciences, Inc, an Affiliate of Merck KGaA, Darmstadt, Germany).

Other described ways to deliver miRNA and/or miRNA mimetics is fromvectors, such as lentiviral constructs, and introducing siRNA moleculesinto cells using electroporation. However, feline FIV lentivirus vectorswhich are based on the feline immunodeficiency virus (FIV) retrovirusand the HIV lentivirus vector system, which is based on the humanimmunodeficiency virus (HIV), carry with them problems related topermanent integration. Electroporation is also useful in the presentinvention, although it is generally only used to deliver siRNAs intocells in vitro.

The target cell types to which miRNA and/or miRNA mimetics can bedelivered using the methods of the invention include eukaryotic cellsincluding, but not limited to hepatocytes, myocytes, neural cells,lipocytes, lymphocytes, macrophages, cardiac cells, endothelial cells,epithelial cells, and the like. In one embodiment, the target cell typeis a tumor cell or a cancer cell including, but not limited to, a lungcancer cell, retinal cancer cell, breast cancer cell, ovarian cancercell, prostate cancer cell, head and neck cancer cell, lymphoma cell,melanoma cell, glioma cell, bladder cancer cell, genital-urinary cancercell, stomach cancer cell, pancreatic cancer cell, liver cancer cell,kidney cancer cell, gastrointestinal cancer and the like. In someembodiments, the target cells are cancer stem cells. In alternativeembodiments, the target cells are selected from the group consisting ofhuman lymphocytes, human dendritic cells, human adult stem cells andembryonic stem cells.

In one embodiment, the nucleic acid encoding a miRNA and/or miRNAmimetics, for example let-7 miRNA or mimetic thereof is present on avector. These vectors include a sequence encoding mature let-7 microRNAand in vivo expression elements. In some embodiments, these vectorsinclude a sequence encoding let-7 pre-miRNA and in vivo expressionelements such that the let-7 pre-miRNA is expressed and processed invivo into a mature let-7 miRNA. In another embodiment, these vectorsinclude a sequence encoding the let-7 pri-miRNA gene and in vivoexpression elements. In this embodiment, the primary transcript is firstprocessed to produce the stem-loop precursor miRNA molecule. Thestem-loop precursor is then processed to produce the mature let-7microRNA.

In some embodiments, the miRNA and/or miRNA mimetics, for example let-7and let-7 mimetics can be delivered in vivo and in vitro. The in vivodelivery as used herein means delivery of the miRNA and/or miRNAmimetic, for example let-7 and let-7 mimetic into a living subject,including human. The in vitro delivery as used herein means delivery ofmiRNA and/or miRNA mimetic, for example let-7 and let-7 mimetic intocells and organs outside a living subject.

Vectors include, but are not limited to, plasmids, cosmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the nucleicacid sequences for producing the microRNA, and free nucleic acidfragments which can be attached to these nucleic acid sequences. Viraland retroviral vectors are a preferred type of vector and include, butare not limited to, nucleic acid sequences from the following viruses:retroviruses, such as: Moloney murine leukemia virus; Murine stem cellvirus, Harvey murine sarcoma virus; marine mammary tumor virus; Roussarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpesviruses; vaccinia viruses; polio viruses; and RNA viruses such as anyretrovirus. One of skill in the art can readily employ other vectorsknown in the art.

Viral vectors are generally based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the nucleic acidsequence of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.

Retroviruses have been approved for human gene therapy trials.Genetically altered retroviral expression vectors have general utilityfor the high efficiency transduction of nucleic acids in viva. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell lined with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W. H. Freeman Co., New York (1990) andMurry, E. J. Ed. “Methods in Molecular L Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

In some embodiments the “in vivo expression elements” are any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient expression of the nucleicacid to produce the microRNA. The in vivo expression element may, forexample, be a mammalian or viral promoter, such as a constitutive orinducible promoter and/or a tissue specific promoter. Examples of whichare well known to one of ordinary skill in the art. Constitutivemammalian promoters include, but are not limited to, polymerasepromoters as well as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase,and beta.-actin. Exemplary viral promoters which function constitutivelyin eukaryotic cells include, but are not limited to, promoters from thesimian virus, papilloma virus, adenovirus, human immunodeficiency virus(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats(LTR) of moloney leukemia virus and other retroviruses, and thethymidine kinase promoter of herpes simplex virus. Other constitutivepromoters are known to those of ordinary skill in the art. Induciblepromoters are expressed in the presence of an inducing agent andinclude, but are not limited to, metal-inducible promoters andsteroid-regulated promoters. For example, the metallothionein promoteris induced to promote transcription in the presence of certain metalions. Other inducible promoters are known to those of ordinary skill inthe art.

Examples of tissue-specific promoters include, but are not limited to,the promoter for creatine kinase, which has been used to directexpression in muscle and cardiac tissue and immunoglobulin heavy orlight chain promoters for expression in B cells. Other tissue specificpromoters include the human smooth muscle alpha-actin promoter.Exemplary tissue-specific expression elements for the liver include butare not limited to HMG-COA reductase promoter, sterol regulatory element1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, humanC-reactive protein (CRP) promoter, human glucokinase promoter,cholesterol L 7-alpha hydroylase (CYP-7) promoter, beta-galactosidasealpha-2,6 sialylkansferase promoter, insulin-like growth factor bindingprotein (IGFBP-1) promoter, aldolase B promoter, human transferrinpromoter, and collagen type I promoter. Exemplary tissue-specificexpression elements for the prostate include but are not limited to theprostatic acid phosphatase (PAP) promoter, prostatic secretory proteinof 94 (PSP 94) promoter, prostate specific antigen complex promoter, andhuman glandular kallikrein gene promoter (hgt-1). Exemplarytissue-specific expression elements for gastric tissue include but arenot limited to the human H+/K+-ATPase alpha subunit promoter. Exemplarytissue-specific expression elements for the pancreas include but are notlimited to pancreatitis associated protein promoter (PAP), elastase 1transcriptional enhancer, pancreas specific amylase and elastaseenhancer promoter, and pancreatic cholesterol esterase gene promoter.Exemplary tissue-specific expression elements for the endometriuminclude, but are not limited to, the uteroglobin promoter. Exemplarytissue-specific expression elements for adrenal cells include, but arenot limited to, cholesterol side-chain cleavage (SCC) promoter.Exemplary tissue-specific expression elements for the general nervoussystem include, but are not limited to, gamma-gamma enolase(neuron-specific enolase, NSE) promoter. Exemplary tissue-specificexpression elements for the brain include, but are not limited to, theneurofilament heavy chain (NF-H) promoter. Exemplary tissue-specificexpression elements for lymphocytes include, but are not limited to, thehuman CGL-1/granzyme B promoter, the terminal deoxy transferase (TdT),lambda 5, VpreB, and lck (lymphocyte specific tyrosine protein kinasep561ck) promoter, the humans CD2 promoter and its 3′ transcriptionalenhancer, and the human NK and T cell specific activation (NKG5)promoter. Exemplary tissue-specific expression elements for the coloninclude, but are not limited to, pp60c-src tyrosine kinase promoter,organ-specific neoantigens (OSNs) promoter, and colon specific antigen-Ppromoter.

In some embodiments, tissue-specific expression elements for breastcells include, but are not limited to, the human alpha-lactalbuminpromoter. Exemplary tissue-specific expression elements for the lunginclude, but are not limited to, the cystic fibrosis transmembraneconductance regulator (CFTR) gene promoter.

Other elements aiding specificity of expression in a tissue of interestcan include secretion leader sequences, enhancers, nuclear localizationsignals, endosmolytic peptides, etc. Preferably, these elements arederived from the tissue of interest to aid specificity. In general, thein vivo expression element shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription. They optionally include enhancer sequencesor upstream activator sequences.

let-7 miRNA or let-7 mimetics, either alone, expressed as a viral vectoror complexed to targeting moieties can be delivered using any deliverysystem such as topical administration, subcutaneous, intramuscular,intraperitoneal, intrathecal and intravenous injections, catheters fordelivering the miRNA and miRNA mimetic, for example let-7 and/or let-7mimetic into, for example, a specific organ, such as breast, brain,liver, heart or kidneys, or into, for example, a specific locationhaving a cancer stem cell, and/or affected with malignant growth orcancer. The let-7 miRNA or let-7 mimetics, either alone or complexed totargeting moieties can also be administered vaginally.

A pharmaceutically acceptable carrier as used herein means anypharmaceutically acceptable means to mix and/or deliver let-7 miRNAand/or let-7 mimetics, either alone or complexed to targeting moietiesto a subject, or in combination with one or more pharmaceuticallyacceptable ingredients.

In the preparation of pharmaceutical formulations containing let-7 miRNAand/or let-7 mimetics, either alone or complexed to targeting moietiesof the present invention in the form of dosage units for oraladministration the compound selected may be mixed with solid, powderedingredients, such as lactose, saccharose, sorbitol, mannitol, starch,arnylopectin, cellulose derivatives, gelatin, or another suitableingredient, as well as with disintegrating agents and lubricating agentssuch as magnesium stearate, calcium stearate, sodium stearyl fumarateand polyethylene glycol waxes. The mixture is then processed intogranules or pressed into tablets.

Soft gelatin capsules may be prepared with capsules containing a mixtureof the active compound or compounds of the invention in vegetable oil,fat, or other suitable vehicle for soft gelatin capsules. Hard gelatincapsules may contain granules of the active compound. Hard gelatincapsules may also contain let-7 miRNA and/or let-7 mimetics, eitheralone or complexed to targeting moieties in combination with solidpowdered ingredients such as lactose, saccharose, sorbitol, mannitol,potato starch, corn starch, arnylopectin, cellulose derivatives orgelatin.

Dosage units for rectal or vaginal administration may be prepared (i) inthe form of suppositories which contain the active substance, i.e. let-7miRNA and/or let-7 mimetics, either alone or complexed to targetingmoieties, mixed with a neutral fat base; (ii) in the form of a gelatinrectal capsule which contains the active substance in a mixture with avegetable oil, paraffin oil or other suitable vehicle for gelatin rectalcapsules; (iii) in the form of a ready-made micro enema; or (iv) in theform of a dry micro enema formulation to be reconstituted in a suitablesolvent just prior to administration.

Liquid preparations for oral administration may be prepared in the formof syrups or suspensions, e.g. solutions or suspensions containing from0.2% to 20% by weight of the active ingredient and the remainderconsisting of sugar or sugar alcohols and a mixture of ethanol, water,glycerol, propylene glycol and polyethylene glycol. If desired, suchliquid preparations may contain coloring agents, flavoring agents,saccharin and carboxymethyl cellulose or other thickening agents. Liquidpreparations for oral administration may also be prepared in the form ofa dry powder to be reconstituted with a suitable solvent prior to use.

Solutions for parenteral administration may be prepared as a solution ofa compound of the invention in a pharmaceutically acceptable solvent,preferably in a concentration from 0.1% to 10% by weight. Thesesolutions may also contain stabilizing ingredients and/or bufferingingredients and are dispensed into unit doses in the form of ampoules orvials. Solutions for parenteral administration may also be prepared as adry preparation to be reconstituted with a suitable solventextemporaneously before use.

The methods of the present invention to also encompass delivery of let-7miRNA and/or let-7 mimetics, either alone or complexed to targetingmoieties orally in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles.

The subject or individual as referred to herein and throughout thespecification includes mammals, such as murine, specifically mice andrats, bovine, and primates, such as human.

Other oral let-7 miRNA and/or let-7 mimetics for use with the inventioninclude solutions or suspensions in aqueous or non-aqueous liquids suchas a syrup, an elixir, or an emulsion. In another set of embodiments,the let-7 miRNA and/or let-7 mimetics may be used to fortify a food or abeverage.

Injections can be e.g., intravenous, intratumoral, intradermal,subcutaneous, intramuscular, or interperitoneal. The composition can beinjected interdermally for treatment or prevention of infectiousdisease, for example. In some embodiments, the injections can be givenat multiple locations. Implantation includes inserting implantable drugdelivery systems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets Inhalation includes administering thecomposition with an aerosol in an inhaler, either alone or attached to acarrier that can be absorbed. For systemic administration, it may bepreferred that the composition is encapsulated in liposomes.

In some embodiments, the miRNA or let-7 mimetic and/or nucleic acidencoding such, for example vectors are provided in a manner whichenables tissue-specific uptake of the agent and/or nucleic acid deliverysystem. Techniques include using tissue or organ localizing devices,such as wound dressings or transdermal delivery systems, using invasivedevices such as vascular or urinary catheters, and using interventionaldevices such as stents having drug delivery capability and configured asexpansive devices or stent grafts.

let-7 miRNA and/or let-7 mimetics of the invention may also be deliveredusing a bioerodible or bioresorbable implant by way of diffusion, ormore preferably, by degradation of the polymeric matrix. Exemplarysynthetic polymers which can be used to form the biodegradable deliverysystem include: polyamides, polycarbonates, polyalkylenes, spolyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, lo hydroxypropyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate), ipoly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene,polyvinylpyrrolidone, and polymers of lactic; acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines and 2 shydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. Examples of non-biodegradable polymersinclude ethylene vinyl acetate, poly(meth)acrylic acid, polyamides,copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) i 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl Imethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In certain embodiments of the invention, the administration of the let-7miRNA and/or let-7 mimetics of the invention may be designed so as toresult in sequential exposures to the composition over a certain timeperiod, for example, hours, days, weeks, months or years. This may beaccomplished, for example, by repeated administrations of let-7 miRNAsand/or let-7 mimetics of the invention by one of the methods describedabove, or by a sustained or controlled release delivery system in whichthe let-7 miRNA and/or let-7 mimetics are delivered over a prolongedperiod without repeated administrations. Administration of the let-7miRNA and/or let-7 mimetics using such a delivery system may be, forexample, by oral dosage forms, bolus injections, transdermal patches orsubcutaneous implants. Maintaining a substantially constantconcentration of the composition may be preferred in some cases.

Other delivery systems suitable for use with the present inventioninclude time release, delayed release, sustained release, or controlledrelease delivery systems. Such systems may avoid repeatedadministrations in many cases, increasing convenience to; the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include, forexample, polymer-based systems such as polylactic and/or polyglycolicacids, polyanhydrides, polycaprolactones, copolyoxalates,polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/orcombinations of these. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Other examples include nonpolymer systems that are lipid-based includingsterols such as cholesterol, cholesterol esters, and fatty acids orneukal fats such as mono-, di and triglycerides; hydrogel releasesystems; liposome-based systems; phospholipid based-systems; silasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; or partially fused implants.Specific examples include, but are not limited to, erosional systems inwhich the composition is contained in a form within a matrix (forexample, as described in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152,4,667,014, 4,748,034 and 29 5,239,660), or diffusional systems in whichan active component controls the release rate I (for example, asdescribed in U.S. Pat. Nos. 3,832,253, 3,854, 480, 5,133,974 and5,407,686). The formulation may be as, for example, microspheres,hydrogels, polymeric reservoirs, cholesterol matrices, or polymericsystems. In some embodiments, s the system may allow sustained orcontrolled release of the composition to occur, for example, throughcontrol of the diffusion or erosion/degradation rate of the formulationcontaining the composition. In addition, a pump-based hardware deliverysystem may be used to deliver one or more embodiments of the invention.

Examples of systems in which release occurs in bursts includes, e. g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by a tonically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional Is systems in which the composition iscontained in a forth within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Examples of systems in which release occurs in bursts includes, e. g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an tonically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in some;embodiments of the invention. “Long-term release,” as used herein, meansthat the implant containing let-7 miRNA and/or let-7 mimetics areconstructed and arranged to deliver therapeutically effective levels ofthe composition for at least 30 or 45 days, and preferably at least 60or days, or even longer in some cases. Long-term release implants arewell known to those of ordinary skill in the art, and include some ofthe release systems described above.

In some embodiments, the let-7 miRNA and/or let-7 mimetics of theinvention may include pharmaceutically acceptable carriers withformulation ingredients such as salts, carriers, buffering agents,emulsifiers, diluents, excipients, chelating agents, fillers, dryingagents, antioxidants, antimicrobials, preservatives, binding agents,bulking agents, silicas, solubilizers, or stabilizers that may be usedwith the active compound. For example, if the formulation is a liquid,the carrier may be a solvent, partial solvent, or non-solvent, and maybe aqueous or organically based. Examples of suitable formulationingredients-30 include diluents such as calcium carbonate, sodiumcarbonate, lactose, kaolin, calcium I phosphate, or sodium phosphate;granulating and disintegrating agents such as corn starch or algenicacid; binding agents such as starch, gelatin or acacia; lubricatingagents such as magnesium stearate, stearic acid, or talc; time-delaymaterials such as glycerol monostearate or glycerol distearate;suspending agents such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone; dispersing or wetting agents such as lecithin orother naturally-occurring phosphatides; thickening agents such as cetylalcohol or beeswax; buffering agents such as acetic acid and saltsthereof, citric acid and salts thereof, boric lo acid and salts thereof,or phosphoric acid and salts thereof; or preservatives such asbenzalkonium chloride, chlorobutanol, parabens, or thimerosal. Suitablecarrier concentrations can be determined by those of ordinary skill inthe art, using no more than routine experimentation. The compositions ofthe invention may be formulated into i preparations in solid,semi-solid, liquid or gaseous forms such as tablets, capsules, elixirs,powders, granules, ointments, solutions, depositories, inhalants orinjectables. Those of ordinary skill in the art will know of othersuitable formulation ingredients, or will be able to ascertain such,using only routine experimentation.

Preparations include sterile aqueous or nonaqueous solutions,suspensions and; emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,injectable organic esters such as ethyl oleate, or fixed oils includingsynthetic mono or all-glycerides. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, 1,3-butandiol, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents and inert gases and the like. Inaddition, so sterile, fixed oils are conventionally employed as asolvent or suspending medium. For i this purpose any bland fixed oil maybe employed including synthetic mono- or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.Those of skill in the art can readily determine the various parametersfor preparing and formulating the compositions of the invention withoutresort to undue experimentation.

In some embodiments, the present invention encompasses let-7 miRNAand/or let-7 mimetics of the invention in association or contact with asuitable carrier, which may constitute one or more accessoryingredients. The final compositions may be prepared by any suitabletechnique, for example, by uniformly and intimately bringing thecomposition into association with a liquid carrier, a finely dividedsolid carrier or both, optionally with one or more formulationingredients as previously described, and then, if necessary, shaping theproduct. In some embodiments, the compositions of the present inventionmay be present as pharmaceutically acceptable salts. The term“pharmaceutically acceptable salts” includes salts of the composition,prepared in combination with, for example, acids or bases, depending onthe particular compounds found within the composition and the treatmentmodality desired. Pharmaceutically acceptable salts can be prepared as;alkaline metal salts, such as lithium, sodium, or potassium salts, or asalkaline earth salts, such as beryllium, magnesium or calcium salts.Examples of suitable bases that may be used to form salts includeammonium, or mineral bases such as sodium hydroxide, lithium hydroxide,potassium hydroxide, calcium hydroxide, magnesium hydroxide, and thelike. Examples of suitable acids that may be used to form salts includeinorganic or mineral acids such as hydrochloric, hydrobromic,hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, phosphorous acids and the like.

Other suitable acids include organic acids, for example, acetic,propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, glucuronic, galacturonic, salicylic, formic,naphthalene-2-sulfonic, and the like. Still other suitable i acidsinclude amino acids such as arginate, aspartate, glutamate, and thelike.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of the miRNA employed andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose is also determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, formulation, or thelike in a particular subject. Therapeutic compositions comprising one ormore nucleic acids are optionally tested in one or more appropriate invitro and/or in viva animal models of disease, to confirm efficacy,tissue metabolism, and to estimate dosages, according to methods wellknown in the art. In particular, dosages can be initially determined byactivity, stability or other suitable measures of treatment vs.non-treatment (e.g., comparison of treated vs. untreated cells or animalmodels), in a relevant assay. Formulations are administered at a ratedetermined by the LD50 of the relevant formulation, and/or observationof any side-effects of the nucleic acids at various concentrations,e.g., as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

In vitro models can be used to determine the effective doses of thenucleic acids as a potential cancer treatment. Suitable in vitro modelsinclude, but are not limited to, proliferation assays of cultured tumorcells, growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described inGiovanella et al., I J. Natl. Can. Inst., 52: 921-30 (1974), mobilityand invasive potential of tumor cells in Boyden Chamber assays asdescribed in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta et al., Intl. J. Dev. Biol., 40:1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

In vivo models are the preferred models to determine the effective dosesof nucleic acids described above as potential cancer treatments.Suitable in vivo models include, but are not limited to, mice that carrya mutation in the KRAS oncogene (Lox-Stop-Lox K-RasGi2D mutants,Kras24TYj) available from the National Cancer Institute (NCI) FrederickMouse Repository. Other mouse models known in the art and that areavailable include but are not limited to models for breast cancer,gastrointestinal cancer, hematopoietic cancer, lung cancer, mammarygland cancer, nervous system cancer, ovarian cancer, prostate cancer,skin cancer, cervical cancer, oral cancer, and sarcoma cancer (seehttp://emice.nci.nih. gov/mouse_models/).

In determining the effective amount of the miRNA and mimetic thereof,for example let-7 and let-7 mimetic to be administered in the treatmentor prophylaxis of disease the physician evaluates circulating plasmalevels, formulation toxicities, and progression of the disease.

The dose administered to a 70 kilogram subject is typically in the rangeequivalent to dosages of currently-used therapeutic antisenseoligonucleotides such as Vikavene (fomivirsen sodium injection) which isapproved by the FDA for treatment of cytomegaloviral RNA, adjusted forthe altered activity or serum half-life of the relevant composition.

In some embodiments, the miRNA and miRNA mimetic, for example let-7miRNA and/or mimetics thereof of the present invention described hereincan supplement the treatment of any known additional therapy, including,but not limited to, antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, and biologic response modifiers. Insome embodiments, additional therapy is, for example, surgery,chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormonetherapy and laser therapy. In some embodiments, the additional therapyis chemotherapy. Two or more combined compounds may be used together orsequentially with miRNAs or miRNA mimetics, for example the let-7 miRNAand/or let-7 mimetics of the present invention. The let-7 miRNA and/orlet-7 mimetics can be administered before the additional therapy, afterthe additional therapy or at the same time as the additional therapy. Insome embodiments, the let-7 miRNA and let-7 mimetics are administered aplurality of times, and in other embodiments, the additional therapiesare also administered a plurality of times.

In some embodiments of the invention miRNA and/or miRNA mimetics, forexample, nucleic acids encoding the let-7 miRNA and let-7 mimetics canalso be administered in therapeutically effective amounts as a portionof an anti-cancer cocktail. An anti-cancer cocktail is a mixture, forexample of a least one let-7 miRNA and/or let-7 mimetic of the presentinvention with one or more additional anti-cancer agents in addition toa pharmaceutically acceptable carrier for delivery. The use ofanti-cancer cocktails as a cancer treatment is routine. Anti-canceragents that are well known in the art and can be used as a treatment incombination with the let-7 miRNA and mimetics thereof as describedherein include, but are not limited to: Actinomycin D,Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophospharnide,Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin,Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium,Etoposide (V16-213), Flosuridine, S-Fluorouracil (5-Fu), Flutamide,Hydroxyurea (hydroxycarb amide), Ifosfamide, Interferon Alpha-2 a,Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog),Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan,Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl,Ockeotide, Paclitaxel; Plicamycin, Procarbazine HCl, Streptozocin,Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate,Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine,Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, andVindesine sulfate, and analogues thereof.

In certain embodiments, the pharmaceutical compositions comprising miRNAand/or miRNA mimetics, for example, pharmaceutical compositionscomprising let-7 miRNA and/or let-7 mimetics can optionally furthercomprise one or more additional therapies or agents. In certainembodiments, the additional agent or agents are anti-cancer agents. Insome embodiments, the therapeutic agents are chemotherapeutic agents,for example cisplatin, paxicital etc. In some embodiments, thetherapeutic agents are radiotherapeutic agents. Examples ofchemotherapeutic agents in the pharmaceutical compositions of thisinvention are, for example nitrogen mustards such as cyclophosphamide,ifosfamide, and melphalan; ethylenimines and methylmelamines such ashexamethylmelamine and thiotepa; pyrimidine analogs such as fluorouraciland fluorodeoxyuridine; vinca alkaloids such as vinblastine;epipodophyllotoxins such as etoposide and teniposide; antibiotics suchas actinomycin D, doxorubicin, bleomycin, and mithramycin; biologicalresponse modifiers such as interferon, platinum coordination complexessuch as cisplatin and carboplatin; estrogens such as diethylstilbestroland ethinyl estradiol; antiandrogens such as flutamine; and gonadotropinreleasing hormone analogs such as leuprolide. Other compounds such asdecarbazine, nitrosoureas, methotrexate, diticene, and procarbazine arealso effective. Of course, other chemotherapeutic agents which are knownto those of ordinary skill in the art can readily be substituted as thislist should not be considered exhaustive or limiting.

In some embodiments, the let-7 miRNA is administered to a subject withother anti-cancer therapies, for example cancer therapies to which thecancer was previously resistant or refractory.

Diseases to be Treated with Let-7 miRNA and Let-7 Mimetics

The invention provides methods for the treatment of any disease ordisorder characterized by lack or reduced expression of let-7. In someembodiments, the disease is cancer. In some embodiments, the cancercomprises cancer stem cells. Cancer treatments promote tumor regressionby inhibiting tumor cell proliferation, inhibiting angiogenesis (growthof new blood vessels that is necessary to support tumor growth) and/orprohibiting metastasis by reducing tumor cell motility or invasiveness.The effect of let-7 miRNA and let-7 mimetics on inhibiting cancer asdisclosed herein and in the Examples is expected to be a general effecton cancer stem cells. That is, where a tumor of any type has a cancerstem cell, one would expect the approach as disclosed herein for usinglet-7 miRNA or let-7 mimetics thereof for the treatment and/orprevention of cancer to work. In particular, the methods andcompositions as disclosed herein are likely to work in a subject thathas any form of cancer where the cancer comprises cancer stem cells witha reduced level of let-7 miRNA as compared to non-stem cancer cells.

The therapeutic formulations described herein comprising let-7 miRNA andlet-7 mimetics may be effective in adult and pediatric oncologyincluding in solid phase tumors/malignancies, locally advanced tumors,human soft tissue sarcomas, metastatic cancer, including lymphaticmetastases, blood cell malignancies including multiple myeloma, acuteand chronic leukemias, and lymphomas, head and neck cancers includingmouth cancer, larynx cancer and thyroid cancer, lung cancers includingsmall cell carcinoma and non-small cell cancers, breast cancersincluding small cell carcinoma and ductal carcinoma, gastrointestinalcancers including esophageal cancer, stomach cancer, colon cancer,colorectal cancer and polyps associated with colorectal neoplasia,pancreatic cancers, liver cancer, urologic cancers including bladdercancer and prostate cancer, malignancies of the female genital tractincluding ovarian carcinoma, uterine (including endometrial) cancers,and solid tumor in the ovarian follicle, kidney cancers including renalcell carcinoma, brain cancers including intrinsic brain tumors,neuroblastoma, askocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers including osteomas,skin cancers including malignant melanoma, tumor progression of humanskin keratinocytes, squamous cell carcinoma, basal cell carcinoma,hemangiopericytoma and Kaposi's sarcoma.

Therapeutic formulations can be administered in therapeuticallyeffective dosages alone or in combination with adjuvant cancer therapysuch as surgery, chemotherapy, radiotherapy, thermotherapy,immunotherapy, hormone therapy and laser therapy, to provide abeneficial effect, e.g. reducing tumor size, slowing rate of tumorgrowth, reducing cell proliferation of the tumor, promoting cancer celldeath, inhibiting angiogenesis, inhibiting metastasis, or otherwiseimproving overall clinical condition, without necessarily eradicatingthe cancer.

Examples of cancers which can be treated by the methods and compositionsas disclosed herein include, but are not limited to, bladder cancer;breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma, AIDSassociated leukemias and adult T-cell leukemia lymphoma; intraepithelialneoplasms including Bowen's disease and Paget's disease, liver cancer;lung cancer including small cell lung cancer and non-small cell lungcancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas;neuroblastomas; oral cancer including squamous cell carcinoma;osteosarcomas; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilm'stumor. In one embodiment, the formulations comprising let-7 miRNA andlet-7 mimetics are administered for treatment or prevention of breastcancer. In another embodiment, the formulations comprising let-7 miRNAand/or let-7 mimetics are administered for treatment or prevention ofbreast cancer.

In some embodiments, the formulations comprising let-7 miRNA and let-7mimetics are administered for treatment or prevention of cancers whichcomprise at least one or a population stem cell cancer cells. Methods toidentify a cancer stem cell are well known in the art, and include, forexample the methods disclosed in U. S Patent Applications; 2008/0020407,2007/0254319, 2007/0244046, 2007/0238127, 2007/0238137, 2007/0248628,2007/0231325, 2007/0134794, and International Patent ApplicationsWO2007147165, WO2007145901, WO2007145840, WO2007142711, WO2007124125,WO2007133250, WO2007118242, WO2007112097, WO2007118238, WO2007053648,WO2003102215, WO2003102215, WO2003050502, and European PatentApplications, EP1726208, EP1697715 and EP1461023, which are allincorporated herein in their entirety by reference. Cancer stem cellswere first detected in the blood cancer, acute myeloid leukemia (AML)(Lapidot et al, Nature 77:645-648 (1994)). More recently it has beendemonstrated that malignant human breast tumors similarly harbor asmall, distinct population of cancer stem cells enriched for the abilityto form tumors in immunodeficient mice. An ESA+, CD44+, CD24−/low,Lin-cell population was found to be 50-fold enriched for tumorigeniccells compared to unfractionated tumor cells (Al-Hajj et al, PNAS700:3983-3988 (2003)).

In addition, therapeutic let-7 miRNA and let-7 mimetics may be used forprophylactic treatment of cancer. There are hereditary conditions and/orenvironmental situations (e.g. exposure to carcinogens) known in the artthat predispose an individual to developing cancers. Under thesecircumstances, it may be beneficial to treat these individuals withtherapeutically effective doses of the nucleic acids encoding let-7miRNA and let-7 mimetics to reduce the risk of developing cancers.

In one embodiment, the pharmaceutical compositions comprising let-7miRNA and/or let-7 mimetics of the present invention are useful to beadministered to a subject who has cancer regression. In anotherembodiment, the pharmaceutical compositions comprising let-7 miRNAand/or let-7 mimetics of the present invention are useful to beadministered to a subject who has a therapy-resistant cancer, forexample a chemotherapy resistant cancer. In some embodiments, thepharmaceutical compositions comprising let-7 miRNA and/or let-7 mimeticsare useful to be administered to a subject who has cancer and has beenexposed to adjuvant cancer therapies.

In another embodiment, the pharmaceutical compositions comprising let-7miRNA and/or let-7 mimetics are useful to be administered to a subjectwith a malignant cancer. In some embodiments, the let-7 miRNA and/orlet-7 mimetics of the present invention can be administered to a subjectwith a cancer or tumor comprising a cancer stem cell.

In one embodiment, the subject is assessed if they are at risk of havinga metastasis or malignant cancer, the method comprising assessing alevel of let-7 in a biological sample, and if the levels of let-7 arebelow a reference level, the subject is at risk of having a metastasisor a malignant cancer. In some embodiments, the biological sample isobtained from a biopsy tissue sample, and in some embodiments, thesample is from a tumor or cancer tissue sample. The level of let-7 canbe determined by methods known by the skilled artisan, for example bynorthern blot analysis or RT-PCR as disclosed in the Examples. In someembodiments, the reference level is the level of let-7 that does notresult in malignancy or a malignant cancer. In some embodiments, thereference level the based on the level of let-7 expression in a normaltissue sample, where in the tissue sample is a biological tissue samplefrom a tissue matched, species matched and age matched biologicalsample. In some embodiments, the reference level is based on a referencesample is from a non-malignant matched tissue sample. In someembodiments, the reference level is based on a reference sample from anon-stem cell cancer tissue sample.

In one embodiment, let-7 miRNA and/or let-7 mimetics in a suitableformulation may be administered to a subject who has a family history ofcancer, or to a subject who has a genetic predisposition for cancer. Inother embodiments, the nucleic acid in a suitable formulation isadministered to a subject who has reached a particular age, or to asubject more likely to get cancer. In yet other embodiments, the nucleicacid in a suitable formulation is administered to subjects who exhibitsymptoms of cancer (e. g., early or advanced). In still otherembodiments, the nucleic acid in a suitable formulation may beadministered to a subject as a preventive measure. In some embodiments,let-7 miRNA and let-7 mimetics in a suitable formulation may beadministered to a subject based on demographics or epidemiologicalstudies, or to a subject in a particular field or career.

Methods to Enrich for Cancer Stem Cells

One aspect of the present invention provides methods for the enrichmentof cancer stem cells. Such methods can provide populations of cancerstem cells for the study of anti-tumor therapies or tumor development orfor screening anti-tumor agents. Such cells can also be useful toidentify miRNAs or other gene products that correlate with or confer“stemness” to such cells (see below). In such an embodiment, the methodscomprise repeated selection for cancer stem cells by sequentialtransplantation of cancer cells and tumor formation under the selectivepressure of low dose cancer therapy. The term “low dose” means a dose ofchemotherapy that is used below the dose which is typically clinicallyadministered as a treatment to eradicate a tumor in a subject. The termlow dose also refers to a dose of the chemotherapy agent which is usedto keep a cancer from reappearing or reoccurring, which is oftenreferred in the clinic as a maintenance dose. In some embodiments, theterm low dose is about half, or less than half, (i.e., <50%, e.g., ≦45%,≦40%, ≦35%, ≦30%, or lower, but generally greater than 1%) (i.e., 50%)the dose which is normally administered clinically to treat or eradicatea tumor in a subject. In some embodiments, the method comprisesobtaining cancer cells and transplanting the cancer cells into a mammal,and administering a low dose cancer therapy to the mammal for a periodof time and sufficient for the cancer cells to develop into a tumor of adesired diameter. In some embodiments, the desired diameter of the tumoris about 2 cm in diameter. Once the desired diameter of the tumor isreached, the tumor is removed from the mammal and dissociated intosingle cells and re-transplanting into another mammal which is alsoadministered a low dose cancer therapy during the formation of the tumorof a desired diameter. The process of tumor formation under low dosecancer therapy and re-transplantation is repeated a plurality of times.In some embodiments, the process is repeated 2,3,4,5 up to 10 times, andin some embodiments the process is repeated more than 10 times. In someembodiments, on the final removal of the tumor from the mammal, thetumor is dissociated into single cells and cultured as embryoid bodies(EBs), herein termed “mammospheres” in the Examples, which comprise apopulation of cells enriched for cancer stem cells.

In some embodiments, the cancer cells are any cancer cells, for examplecancer cell lines or primary cancer cells obtained from a subject. Insome embodiments, the cancer cells are human cancer cells. Inalternative embodiments, the cancer cells are mammalian, for examplerodent. In some embodiments, the cancer cell is a breast cancer cell,and in other embodiments the cancer cells can be from breast cancer,lung cancer, head and neck cancer, bladder cancer, stomach cancer,cancer of the nervous system, bone cancer, bone marrow cancer, braincancer, colon cancer, esophageal cancer, endometrial cancer,gastrointestinal cancer, genital-urinary cancer, stomach cancer,lymphomas, melanoma, glioma, bladder cancer, pancreatic cancer, gumcancer, kidney cancer, retinal cancer, liver cancer, nasopharynx cancer,ovarian cancer, oral cancers, bladder cancer, hematological neoplasms,follicular lymphoma, cervical cancer, multiple myeloma, osteosarcomas,thyroid cancer, prostate cancer, colon cancer, prostate cancer, skincancer, stomach cancer, testis cancer, tongue cancer, or uterine cancer.

In some embodiments, the cancer cells are transplanted into the mammalat the location most suitable for the specific cancer cell. For example,breast cancer cells are implanted in the mammary fat pad. In alternativeembodiments, the cancer cells are implanted into brain where the cancercells are obtained from brain cancer.

In some embodiments, the mammal used in the transplantation is a rodent,and in some embodiments the rodent is a genetically modified rodent, andin some embodiments the rodent is an immunocompromised rodent, forexample but not limited to a NOD/SCID mouse.

In some embodiments the cancer therapy is chemotherapy, radiotherapy,thermotherapy, immunotherapy, hormone therapy and laser therapy. In someembodiments, more than one cancer therapy can be administered, and insome embodiments the cancer therapies can be administered at the sametime or sequentially. In some embodiments, the cancer therapies can beof different types, for example one cancer therapy can be achemotherapeutic agent and one cancer therapy is a immunotherapy, and inalternative embodiments the cancer therapies can be of the same types,for example two or more different chemotherapeutic agents. In someembodiments, the cancer therapies can be administered to the same mammaland in anther embodiment they can be administered to different mammals.

In some embodiments, administration is performed by any means and at anyfrequency intervals for sustained cancer therapy. For example, in someembodiments, the administration is continuous administration, and insome embodiments administration is, for example but not limited to twicea day, every day, every other day, twice a week, once a week, everyother week or once a month. In some embodiments, administration isintravenous, intradermal, intramuscular, intraarterial, interlesional,percutaneous, subcutaneous, intraperitoneal or by aerosol.

The in vivo delivery as used herein means delivery of the miRNA and/ormiRNA mimetic, for example let-7 and let-7 mimetic into a livingsubject, including human. The in vitro delivery as used herein meansdelivery of miRNA and/or miRNA mimetic, for example let-7 and let-7mimetic into cells and organs outside a living subject.

In another embodiment, the present invention provides methods toidentify miRNA that contribute to the self-proliferative capacity and/ortumorogenicity of cancer stem cells. The method comprises comparing themiRNA expression profile of a cancer stem cell, for example a cancerstem cell which has been enriched by the methods described herein, withthe miRNA expression profile of a reference sample. In some embodiments,a reference is any biological sample. A difference in the level ofexpression of a miRNA in the cancer stem cell sample as compared withthe level of expression of the same miRNA in a reference sampleidentifies that the miRNA contributes to, in whole or in part, to theself-proliferative capacity and/or tumorogenicity of the cancer stemcell. In this embodiment, the difference in the level of expression ofthe miRNA in the cancer stem cell as compared with the reference levelis a statistically significant change, but generally is at least about10%, for example, at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80% or at least about 90%, or at leastabout 100%, or at least about 2-fold, or at least about 2.5-fold, or atleast about 3 fold, or at least about 5-fold, or at least about 10-foldor more, or any integer in between a 10% difference and a 10-folddifference or more.

In some embodiments, the reference sample is a plurality of cancercells, and in some embodiments the cancer cell is a cancer cell fromwhich the cancer stem cells was derived using the methods of the presentinvention or from a cell derived from the cancer stem cell, for examplea differentiated cancer stem cell. A number of miRNA profiles fromreference samples can be compared to the miRNA profile of the cancerstem cell.

In further embodiments, the present invention provides methods to assessthe level of contribution of the miRNA identified to contribute to thecancer stem cell self-proliferative capability, the method comprisingeither introducing or inhibiting the miRNA in the cancer stem cell,depending if the miRNA being assessed is downregulated or upregulatedrespectively, in the cancer stem cell as compared with the referencesample.

In some embodiments, a cancer stem cell's self-proliferative capacity isdetermined by the ability of the cancer stem cell to form embryoidbodies (EBs) (or mammospheres) in culture. To assess the effect of themiRNA on the self-proliferative ability of the cancer stem cell, if themiRNA is downregulated in the cancer stem cell compared to the referencesample, and the miRNA or mimetic thereof is introduced back into thecancer stem cell and the ability of the cancer stem cell to formmammospheres or EBs in culture is reduced, the miRNA contributes to theself-proliferative ability and/or tumorogenicity of the cancer stemcell. Similarly, if the miRNA is upregulated in the cancer stem cell ascompared to the reference sample, and the expression and/or activity ofthe miRNA is inhibited in cancer stem cell and the ability of the cancerstem cell to form mammospheres or EBs in culture is reduced, the miRNAcontributes to the self-proliferative ability and/or tumorogenicity ofthe cancer stem cell.

EXAMPLES

Throughout this application, various publications are referenced. Thedisclosures of all of the publications and those references cited withinthose publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. The followingexamples are not intended to limit the scope of the claims to theinvention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Methods

Primary Tumor Specimens.

Tumors were obtained following a protocol approved by the ethicscommittee of the No. 2 Affiliated Hospital of Sun-Yat-Sen University inChina from 11 consented female patients (median age 52 years) withbiopsy-diagnosed poorly differentiated invasive ductal carcinomas of thebreast. Five patients received 4 cycles of neoadjuvant chemotherapy with5-fluorouracil 500 mg/m², epirubicin 100 mg/m² and cyclophosphamide 500mg/m² followed by modified radical resection of the breasts. Modifiedradical resection was performed in 6 cases without neoadjuvantchemotherapy. The average tumor size was 3.4 cm (range, 2.5-4.3 cm), andall patients had axillary lymph node metastasis. Surgical specimens werereceived in the laboratory within 20 min of surgery, and were immediatemechanically disaggregated and digested with collagenase as described¹⁰.Single cancer cells were obtained by filtration through a 30μ filter.

In-Vivo Enrichment of Breast Tumor-Initiating Cells.

Female NOD/LtSz-scid/scid (NOD/SCID) mice were bred and maintained underdefined conditions at the Animal Experiment Center of Sun-Yat-SenUniversity, and all animal experiments were approved by the Animal Careand Use Committee of the Sun-Yat-Sen University. SKBR3 cells (ATCC) werepassaged in NOD/SCID mice by injecting 2×10⁶ cells into the mammary fatpad of 5-week-old mice. To select for chemoresistant breasttumor-initiating cells, Epirubicin (8 mg/kg, Pharmacia and Upjohn) wasinjected into the tail vein weekly. Single cell suspensions wereobtained by collagenase digestion as described¹⁰ of tumor xenografts,removed when tumors reached ˜2 cm in diameter. The dissociated cellswere repetitively passaged in Epirubicin-treated NOD/SCID mice as abovefor 3 generations. Freshly isolated single tumor cells obtained from the3^(rd)-generation xenografts (SK-3rd) were used to generate mammospherecultures.

Mammosphere Culture.

SK-3rd and SKBR3 cells were cultured in suspension at a clonal densityof 1,000 cells/mL in serum-free DMEM-F12 (Bio Whittaker), supplementedwith B27 (1:50, Invitrogen), 20 ng/mL EGF (BD Biosciences), 0.4% bovineserum albumin (Sigma) and 4 μg/mL insulin (Sigma)²⁵. Alternatively,single-cell suspension culture was obtained by suspending a singleSK-3rd or SKBR3 cell in 200 μL of the above medium in 96-well plates. Topropagate mammospheres in vitro, the spheres were collected by gentlecentrifugation and were dissociated to obtain single cells enzymaticallyand mechanically as described²⁵. Single cells were then cultured insuspension to generate mammospheres of the next generation. Thepercentage of wells with mammospheres was analyzed at indicated times,and the size of mammospheres (cells/sphere) was determined bydissociating and counting the cells in the spheres.

Generation of microRNA and shRNA-Expressing Lentiviruses.

Oligonucleotides encoding let-7a1 pre-miRNA³³ (SEQ ID NO: 7) or shRNAtargeting H-RAS1⁴⁶ or eGFP were synthesized according to previouslypublished sequences and cloned under the control of the U6 promoter inthe lentiviral vector lentilox pLL3.7 as previously reported⁴⁷.Generation of lentivirus vectors was performed as described⁴⁷ byco-transfecting pLL3.7 carrying the miRNA or shRNA expression cassettewith helper plasmid pCMV-VSV-G and pHR′8.9ΔVPR in 293 T cells usingFuGENE 6 (Roche). The viral supernatant was collected 48 hrs aftertransfection, and viral titers determined by transducing HeLa cells atserial dilutions and analyzing GFP expression by flow cytometry.

Transduction with Lentivirus Vectors.

SK-3rd cells dissociated from the primary mammospheres werespin-infected with 1 mL of lentiviral supernatant containing 8 μg/mLpolybrene for 2 hr at a multiplicity of infection (MOI) of 1:5, followedby incubation for 2 hours at 37° C. Transduction efficiency, evaluatedby GFP expression, was >90%.

Transfection with Let-7a ASO.

After washing in D-MEM medium without serum, SKBR3 cells weretransfected in 24-well plates with 30 pmol of let-7 ASO or 30 pmol of acontrol lin-4 ASO (Ambion) using Lipofectamine 2000 overnight. Cellswere harvested for further experiments 48 hr post-transfection.

miRNA Microarray Analysis.

Total RNA enriched for small RNAs was isolated using the mirVana RNAIsolation Kit (Ambion), and miRNAs were then excised from RNAelectrophoresed through polyacrylamide gels. A poly(A) tail was appendedto the 3′-end of miRNAs from all the above samples with a mixture ofunmodified and amine-modified nucleotides (Ambion). The tailed sampleswere fluorescently labeled using an amine-reactive Cy3 dye (Amersham),and the unincorporated dyes were removed with glass fiber filters. Thesamples were hybridized for 14 hr onto slides arrayed with miRNA probesfrom the NCode™ miRNA Microarray Probe Set (Invitrogen). Slides werethen washed 3×2 min in 2×SSC and scanned using a Generation β arrayscanner (Amersham Pharmacia). Fluorescence intensities for theCy3-labeled samples were normalized by the median total Cy3 signal onthe arrays. The signal intensity of each element was analyzed usingArrayVision (Imaging Research Ltd), and images were created with DMVS2.0 software (Chipscreen Biosciences Ltd).

Northern Blot.

Northern blot for let-7 miRNA was performed as previously reported³³.Briefly, 10 μg of RNA were fractionated on a 15% denaturingpolyacrylamide gel. The RNA was then electrotransferred to Nytran Plus(Schleicher & Schuell, Inc., Keene, N. H.) at 200 mA for 2.5 hr, UVcross-linked at 1,200 μF, and prehybridized for 30 min at 40° C. inUltraHyb buffer (Ambion). The let-7 probe(5′-TACTATACAACCTACTACCTCAATTTGCC; SEQ ID NO:12) was radiolabeled asdescribed³³ and blots were hybridized in 10 mL of UltraHyb buffer(Ambion). After washing 2×5 min at room temperature in 2×SSC, 0.1% SDSand 3×10 min in 1×SSC, 0.1% SDS, the blots were analyzed on aphosphorimager (Molecular Dynamics). The process was repeated using aradiolabeled probe for U6 snRNA (5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG; SEQID NO:13)⁴².

Quantitative RT-PCR.

Real-time reverse transcription PCR was performed using an ABI Prism7900 Sequence Detection System (Perkin-Elmer Applied Biosystems). SYBRgreen (Molecular Probes) was used to detect PCR products. All reactionswere done in a 25-μl reaction volume in triplicate. Primers for maturelet-7a miRNA (Probe 1: SEQ ID NO:14 and SEQ ID NO:15) and U6 snRNA werefrom Ambion. PCR amplification consisted of 10 min of an initialdenaturation step at 95° C., followed by 55 cycles of PCR at 95° C. for30 s, 56° C. for 30 s and 72° C. for 15 s. Standard curves weregenerated and the relative amount of target gene mRNA was normalized toU6 snRNA. Specificity was verified by melt curve analysis and agarosegel electrophoresis. To quantify cancer metastasis in mouse lungs andlivers, qRT-PCR for human hypoxanthine-guanine-phosphoribosyltransferase(hHPRT), was performed on Trizol (Life Technologies, Gaithersburg,Md.)-isolated total RNA using described primers for human HPRT (hHPRT)and mouse GAPDH (mGAPDH)⁴⁸. Following reverse transcription for 30 minat 48° C. and Taq activation for 10 min at 95° C., 40 cycles of PCR at95° C. for 20 sec, 55° C. for 30 sec, and 72° C. for 30 sec wereperformed.

Let-7 Luciferase Assay.

To evaluate the miRNA function of let-7, a pMIR-REPORT™ luciferasereporter vector with a let-7 target sequence (SEQ ID NO: 9) (as well asSEQ ID NO:10 and SEQ ID NO:11) was cloned into its 3′UTR (luc-let-7-ts)(Ambion) was used. The reporter vector plasmid was transfected usingLipofectamine 2000 according to the manufacturer's instruction. Tocorrect for transfection efficiency, a luciferase reporter vectorwithout a let-7 target was transfected in parallel. Luciferase activitywas assayed by luciferase assay kit (Promega). let-7 miRNA function wasexpressed as percentage reduction in the luciferase activity of cellstransfected with the reporter vector containing the let-7 targetsequence compared to cells transfected with the vector without the let-7target.

Western Blot.

Protein extracts were resolved through 12% SDS-PAGE, transferred tonitrocellulose membranes, probed with rabbit polyclonal antibodiesagainst human H-RAS (Upstate) or human Oct-4 (Chemicon Int), and thenwith peroxidase-conjugated goat anti-rabbit Ig secondary antibody(Oncogene Research Product), and then visualized by chemiluminescence(Amersham).

Flow Cytometry.

For cell surface staining, unfixed cells were incubated withFITC-labeled anti-CD44 and PE-labeled anti-CD24 (PharMingen) at 4° C.For staining cytoplasmic antigens, cells were permeabilized with theCaltag Laboratories (Burlingame) Fix and Perm kit and stained withFITC-labeled CK14 and PE-labeled CK18 (Neomarkers). Cells were analyzedby flow cytometry on a FACScalibur instrument with CellQuest software(BD).

Cell Proliferation.

³H-thymidine (1 μCi) was added for 6 h to 2×10⁴ cells in octuplicatemicrotiter wells, before harvesting and analysis by scintillationcounting using a Top Count microplate reader (Packard).

Tumor Implantation.

Indicated numbers of SKBR3 or SK-3rd cells dissociated from mammosphereswere injected subcutaneously into the mammary fat pads of 5-week-oldNOD/SCID mice. Mice were examined by palpation for tumor formation forup to 60 d. After tumors were detected, tumor size was measured every 3d by calipers, and tumor volumes calculated as Volume (mm³)=L×W²×0.4.Mice were sacrificed by cervical dislocation and the presence of tumorswas confirmed by necropsy. Tumor xenografts as well as whole lung andliver tissues were harvested, weighed and snap-frozen in liquidnitrogen. Portions of the lung and liver tissues were used for real-timeRT-PCR for human HPRT to evaluate metastasis. Cryosections (4 μm) werestained with hematoxylin and eosin and used for immunohistochemistry.

Immunohistochemistry.

Cryosections were stained using anti-human RAS (Upstate) or anti-humanPCNA (BD Biosciences) mAb. Briefly, endogenous peroxidase activity wasquenched by incubation with 3% hydrogen peroxide in methanol for 5 min.Sections were washed in phosphate buffered saline (PBS) and blocked for1 h in a washing buffer containing 5% normal goat serum (Sigma ChemicalCo. St Louis, Mo.). The primary antibody was added for incubationovernight at 4° C. After washing in PBS, slides were incubated withbiotinylated goat anti-mouse Ig and then with streptavidin conjugatedwith horseradish peroxidase. After further washing in PBS, slides weredeveloped with diaminobenzidine (DAB; Dako Corp. Carpinteria, Calif.)lightly counterstained with hematoxylin. Negative control slides werestained with isotype mouse immunoglobulin to replace primary antibodies.The percentage of H-RAS and PCNA-positive tumor cells was calculated bycounting 1,000 tumor cells.

Statistics.

All in vitro experiments were performed either in triplicate or inpentuplicate. The results are described as mean±SD. Statistical analysiswas performed by one-way analysis of variance (ANOVA) and comparisonsamong groups were performed by independent sample t-test or Bonferroni'smultiple-comparison t-test.

Example 1 Low-Dose Chemotherapy Selects for Tumor-Initiating BreastCancer Cells

Resistance to chemotherapy distinguishes tumor-initiating cells fromother cancer cells^(1,2,17). Accordingly, treatment with chemotherapyshould enrich for tumor stem cells compared to more differentiatedprogeny. To examine this, the inventors compared the proportion ofself-renewing cancer cells in primary breast cancer tissues surgicallyremoved from patients with poorly differentiated invasive breast cancerwho received four cycles of preoperative chemotherapy with tumors frommatched untreated patients. Freshly isolated cells were cultured insuspension in medium supplemented with epidermal growth factor (EGF),B27 and insulin to generate mammospheres (or emboid bodies), apreviously described method for culturing both mammary gland progenitorcells²⁵ and breast tumor-initiating cells¹⁰. The self-renewal potentialof breast tumor-initiating cells can be gauged by their capacity to giverise to mammospheres¹°. From 5 patients who received neoadjuvantchemotherapy, 5.8±2.6% of tumor cells formed mammospheres after 15 d ascompared with 0.4±0.3% from 6 chemotherapy-naïve patients, a 14-foldincrease (P<0.001, FIG. 1 a). Furthermore, the primary mammospheres fromneoadjuvant chemotherapy patients could be passaged for at least 8-10generations (end point of the study), while those from patients withoutchemotherapy vanished within 2-3 generations. Self-renewing breastcancer cells have been shown to be CD44⁺CD24^(−9,10,15); 70±8% offreshly examined primary tumor cells from chemotherapy-treated patientshad this phenotype, while only 9±3% of cells from untreated patients did(P<0.001, FIG. 1 b). These data demonstrate that neoadjuvantchemotherapies selectively enhance the proportionate survival of breasttumor-initiating cells in primary cancer tissues.

The inventors took advantage of this finding to demonstrate the abilityto enrich for tumor-initiating cells by consecutively passaging breastcancer cells in NOD/SCID mice treated with low dose chemotherapy. Miceinjected in the mammary fat pad with SKBR3 tumor cells were treated withEpirubicin weekly for 10-12 weeks until xenografts reached a diameter of˜2 cm. Cells from the 3^(rd) passaged xenograft (SK-3rd) were culturedin suspension to generate mammospheres. The number of mammospheresreflects the quantity of stem cells with self-renewal potential, whilethe number of cells per mammosphere measures the self-renewal capacityof each cell^(25,26). The inventors assessed the percentage ofmammospheres formed by SK-3rd and their parental SKBR3 counterpartsafter 15 d in suspension culture. Mammosphere formation in SK-3rd wasapproximately 20-fold higher than SKBR3 (16.3% vs. 0.8%, P<0.001, FIG. 1c). Moreover, dissociated SK-3rd cells from primary mammospheresgenerated an equivalent proportion of secondary spheres and subsequentlytertiary spheres (FIG. 1 c), demonstrating their self-renewing potentialin vitro. Long-term SK-3rd mammosphere cultures could be maintainedfor >50 passages, while within 3-4 passages, mammospheres from SKBR3failed to generate secondary spheres and became adherent anddifferentiated. These findings were confirmed by single cell cloning(FIG. 6). SK-3rd mammospheres were observed beginning at day 5 andincreased in size and cell number until day 15 (FIG. 1 d). Secondarymammospheres could be passaged >40 times from single cell SK-3^(rd)clones. However, mammospheres did not appear until d 15 in parentalSKBR3 cells and were about 18-fold fewer in number and much smaller(FIG. 1 d).

Dissociated SK-3rd mammospheres could be differentiated in vitro byplating on collagen in serum-containing medium lacking exogenous growthfactors. Within 24 hr, the suspended cells began to adhere and spreadand could thereafter be maintained and expanded as differentiated cells(FIG. 1 e). Moreover, 93% of mammosphere-derived SK-3rd, but fewer than0.5% of parental SKBR3, were CD44⁺CD24⁻, the phenotype oftumor-initiating breast cancer cells^(9,10,15) (FIG. 1 g). During invitro differentiation of SK-3rd, the percentage of CD44⁺CD24^(−/low)cells decreased steadily to ˜32% on d 3, 11% on d 7 and 2% on d 10.Furthermore, SK-3rd, but not SKBR3, cells highly expressed the stemcell-associated transcription factor Oct-4, which declined upon in vitrodifferentiation (FIG. 1 f). Therefore SK-3rd mammospheric cells have theself-renewing and differentiating capability and phenotypic propertiesexpected of breast cancer stem cells.

Example 2 Breast Tumor-Initiating Cells have Reduced Expression of Let-7miRNAs and Let-7 Homologues or Let-7 Mimetics

The inventors used miRNA microarrays to compare miRNA expression inmammosphere-derived SK-3rd with their in vitro differentiated progenyand the parental SKBR3 cells. As has been reported for ES cells^(22,27),most of the 52 miRNAs that were reproducibly expressed above backgroundin any of the 3 cell lines had reduced expression in SK-3rd comparedwith either the differentiated SK-3rd cells or SKBR3 (FIG. 2 a). Clusteranalysis of multiple samples showed a clear distinction betweenmammospheric cells and the other two adherent lines (data not shown).Using ANOVA analysis on the normalized chip data, we identified a numberof human miRNAs whose expression in mammospheric cells was significantlydifferent from the differentiated and parent cells. Among them, thelet-7 family emerged as the most consistently and significantly reducedmiRNAs. let-7 was initially identified as a miRNA that regulatesdevelopment in C. elegans ²⁸, where it was shown to target key genesinclude lin-4I, hbl, daf-12, pha-4 and let-60 (a RAS homolog)²⁹⁻³¹. Inhumans, 11 homologues of let-7 miRNAs exists, which are differentiallyexpressed in different tissues, but are believed to have redundanttargets and functions^(29,32). Human let-7, which is downregulated insome cancers and associated with poor prognosis in lung cancer³³,targets the RAS oncogene and thereby acts as a tumor suppressor³¹. Toverify the reduction of let-7 miRNAs in SK-3rd, the inventors performedNorthern blot using a probe that recognizes a variety of let-7 familymembers or homologues³¹ (FIG. 2 b). let-7 was barely detected in SK-3rd,but increased with differentiation and was abundant in the parent SKBR3cells. This result was verified using a let-7a-specific primer forquantitative reverse transcription-PCR (qRT-PCR). let-7a was 10-foldlower in SK-3rd than differentiated SK-3rd, and the level in thedifferentiated cells was comparable to SKBR3 (FIG. 2 c).

Example 3 Let-7 Activity is Low in Breast Cancer Stem Cells andIncreases During Differentiation

To investigate let-7 function, the inventors transfected a luciferasereporter vector containing a let-7 target sequence in its 3′UTR intoSK-3rd, differentiated SK-3rd and SKBR3. Luciferase activity wassuppressed by 52% in differentiated SK-3rd cells (P<0.001) and by 78% inSKBR3 (P<0.001), while there was no suppression in SK-3rd (FIG. 2 d).Infection of SK-3rd with a lentivirus expressing let-7a pre-miRNAenhanced miRNA expression and function comparably to that of thedifferentiated progeny cells (FIG. 2 b-d). Co-transfection ofdifferentiated SK-3rd cells, SKBR3 or let-7a lentivirus-infected SK-3rdwith a let-7 antisense oligonucleotide (ASO) significantly reduced thesuppression in luciferase activity mediated by endogenous or exogenouslet-7 (P<0.01; FIG. 2 d).

Since RAS is a major target of let-7 miRNAs³¹, the inventors nextcompared HRAS 1 mRNA and protein expression in the 3 cell lines. H-RASprotein was highly expressed in SK-3rd stem cells, but was greatlyreduced in differentiated SK-3rd cells and SKBR3. (Other RAS proteinswere not detected in any of these cells (data not shown). As expected,introduction of let-7a or RAS-shRNA by lentiviruses into SK-3rd reducedH-RAS protein to the level found in the differentiated cells, whileinhibiting let-7 with a specific ASO in the parent SKBR3 cellsup-regulated H-RAS expression substantially (FIG. 2 e). However, HRAS1mRNA, measured by qRT-PCR, did not differ significantly amongst the 3sources of cells (data not shown). Therefore, let-7 silences RASexpression by inhibiting translation, and reduced let-7 in breast cancerstem cells leads to RAS over-expression.

Example 4 Let-7 is Reduced in Breast Tumor-Initiating Cells from PrimaryCancers

To confirm that the results with the breast cancer cell line arephysiologically relevant to primary breast cancers, the inventorsexamined let-7 family expression in the breast tumor-initiating cellsfrom primary cancers by Northern blot and quantified it by qRT-PCR usinga let-7a-specific primer (FIG. 2 f,g). In agreement with the data fromSK-3rd cells, the tumor-initiating cells from primary mammospheres fromprimary cancers from chemotherapy-treated patients had reduced let-7(Fog 20, for example at least about, 20%, or at least about 30% or atleast about 50% lower level, as compared with the primary cancer cellsfreshly isolated from tissue samples of untreated patients. When thetumor-initiating mammospheric cells were differentiated for 14 d inadherent cultures, let-7 expression returned to the level inuntreated-patient cells.

Example 5 Reduced Let-7 is Required to Maintain Self-Renewal ofTumor-Initiating Cells

To test the importance of low let-7 expression in breast cancer stemcells, the inventors first studied the effect of enforced let-7aexpression in SK-3rd on self-renewal using the mammosphere-formingassay. As disclosed above, the ability of a cell to form a mammosphere(i.e. an embryoid body) indicates the self-renewal capacity of the cell.SK-3rd cells infected with let-7a lentivirus formed 5.3-fold fewersecondary mammospheres than uninfected SK-3rd or SK-3rd cells infectedwith lentiviral vectors that were empty or expressed an eGFP-shRNA (FIG.3 a). Mammosphere formation was also delayed and the mammospheres thatformed were 2-3-fold smaller in let-7a-expressing SK-3rd cells comparedwith control SK-3rd cells (FIG. 3 b). Importantly, the let-7a-transducedmammospheric cells could only be passaged for 8-10 generations.Therefore let-7a transduction not only reduced the number oftumor-initiating cells, but also weakened their self-renewing capacity.Conversely, transfecting let-7 ASO into parental SKBR3 cells enhancedtheir ability to form mammospheres by ˜6-fold (FIG. 3 c).

Example 6 Reduced Let-7 Maintains Tumor-Initiating Cell Proliferation,but Inhibits their Differentiation

Another important stem cell property is the potential to proliferateduring differentiation. When mammospheric SK-3rd cells were plated fordifferentiation, let-7 expression measured by qRT-PCR, increasedgradually and plateaued on d 6 (FIG. 3 d). Resting SK-3rd cellsproliferated at half the rate of parental SBR3 cells as measured by [³H]incorporation (FIG. 3 e). During differentiation, SK-3rd proliferationincreased about 7-fold from baseline to a peak on d 4 and then fell by d8 to a level somewhat higher than that of the parental cell line(P<0.01). To investigate the effect of let-7 on proliferation, theinventors measured proliferation during differentiation of SK-3rd cellstransduced with pre-let-7a. Enhancing let-7a expression reduced peak[³H]-incorporation by 58%, demonstrating that reduced let-7 enhances theproliferative potential of differentiating stem cells.

Another hallmark of stem cells is their undifferentiated state andpotential to differentiate into multiple lineages. Mammospheric SK-3rdcells expressed neither myoepithelial (CK14) nor luminal epithelial(CK18) cytokeratins (data not shown), while the parental SKBR3 cellswere 70% myoepithelial and 30% luminal epithelial (FIG. 3 f). However,after 10 days of differentiation, most of the SK-3rd cells expresseddifferentiation markers (44±4% CK14+CK18−, 28±7% CK14-CK18+), but 15±3%were lin⁻. let-7a over-expression significantly (P<0.001) reduced theproportion of lin⁻ cells to 6±2%, but control lentiviruses, including alentivirus expressing RAS-shRNA (see below), had no effect on in vitrodifferentiation. Taken together, these data demonstrate that low let-7expression helps to maintain the undifferentiated status andproliferative potential of breast tumor-initiating cells.

Example 7 Let-7 Expression Silences RAS and Other Genes Silencing RASOnly Partly Recapitulates the Effects of Let-7 Expression

Since RAS is a well-documented target of let-7³¹ the inventorsinvestigated whether the effects of reduced let-7 in maintainingtumor-initiating properties of SK-3^(rd) could be attributed to RASoncogene expression. A lentivirus expressing RAS-shRNA reduced H-RASprotein in SK-3rd to the level in SKBR3 or differentiated SK-3rd andcomparably to that of the let-7a-lentivirus (FIG. 2 e). SK-3rd cellswith silenced H-RAS formed mammospheres at a level that was about halfthat of untransduced or control vector transduced SK-3^(rd) cells, butabout 3-fold greater than cells transfected with let-7a-lentivirus (FIG.3 a); moreover the mammospheres that formed were intermediary in size(465±94 cells vs. 745±155 cells for untransduced SK-3rd and 277±82 cellsfor let-7a-transduced SK-3rd on d 20; FIG. 3 b). Silencing RAS alsosomewhat reduced SK-3^(rd) proliferation under differentiatingconditions, but much less than expressing let-7a (FIG. 3 e, P<0.001, ond 4 of differentiation, the peak of proliferation). As noted above,silencing RAS, unlike over-expressing let-7a, in SK-3rd did not reducethe residual proportion of undifferentiated cells lacking cytokeratinexpression after in vitro differentiation (FIG. 3 f). Therefore, let-7silencing of RAS explains some, but not all, of the role of let-7 inconverting breast cancer stem cells to more differentiated progeny.These data demonstrate that expression of let-7 targets other RNAtranscripts in addition to RAS in breast cancer stem cells whichcontribute to the “stemness” or self-renewal capacity of cancer stemcells, such as breast cancer stem cells.

Example 8 Lack of Let-7 Facilitates Tumorigenesis of BreastTumor-Initiating Cells

Cancer stem cells establish tumor xenografts much more readily thandifferentiated tumor cells^(1,2). When mammospheric SK-3rd cells wereinoculated subcutaneously into NOD/SCID mice, eight of ten miceengrafted with 2×10³ cells generated tumors that were first detected36-45 d later (Table 1, FIG. 4 a). All animals injected with 10 or100-fold more cells developed tumors within 30 and 21 days,respectively. Within 14 days after tumors were identified, their sizesreached 1.8±0.7 cm in diameter. By contrast, no mice inoculated with2×10³ or 2×10⁴ SKBR3 cells developed tumors by day 60, while tumorsdeveloped by day 45 in only 3 of 10 animals inoculated with 2×10⁵ SKBR3cells. Therefore, SK-3rd cells are at least 100-fold more tumorigenicthan the parental cell line. When let-7a expression was enforced inmammospheric SK-3rd cells, tumors developed in only 20%, 50% and 70% ofmice inoculated with 2×10³, 2×10⁴ and 2×10⁵ cells, respectively.Moreover, the let-7a-expressing tumors grew more slowly than theuntransduced or control vector-transduced SK-3rd tumors; it took 25-33days for the tumors after they became palpable to reach 2.0 cm indiameter, while the control SK-3rd cells reached that size in ˜12 d(FIG. 4 a).

TABLE 1 Incidence of tumors and metastasis in mammospheric SK-3^(rd)cells and SKBR3 cells in NOD/SCID mice. 1 × 10³ 1 × 10⁴ 1 × 10⁵ LungLiver Lung Liver Lung Liver Number of cells inoculated Tumors metastasismetastasis Tumors metastasis metastasis Tumors metastasis metastasisMammospheric Untransduced 8/10 6/10 3/10 10/10 7/10 4/10 10/10 8/10 6/10SK-3^(rd) cells Lentivirus 8/10 5/10 3/10 10/10 8/10 5/10 10/10 8/105/10 Lenti-let-7  2/10* 1/10 0/10  5/10* 3/10 1/10  7/10 4/10 3/10RAS-shRNA  3/10* 2/10 1/10  7/10 5/10 3/10 10/10 7/10 4/10 SKBR3  0/10^(∞)  0/10* 0/10   0/10^(#)   0/10^(∞) 0/10  3/10 0/10 0/10 *= p <0.05; ^(∞)= p < 0.01, ^(#)= p < 0.001 compared with untransducedmammospheric SK-3^(rd) cells.

Furthermore, primary mammospheres from chemotherapy patients could bepassaged for at least eight to ten generations (endpoint of the study),while those from patients without chemotherapy vanished within two tothree generations. In the primary breast cancers; 74%±7% of tumor cellsfrom chemotherapy-treated patients, but only 9%±4% of cells fromuntreated patients, were CD44⁺CD24⁻/low, the phenotype ascribed to BT-IC(Al-Hajj et al., 2003; Ponti et al., 2005) (p<0.001, FIG. 1B).Enrichment of BT-IC by chemotherapy was confirmed by studying pairedspecimens from seven patients obtained by biopsy prior to chemotherapyand at surgery following neoadjuvant chemotherapy. Only 0.5%±0.3% oftumor cells before chemotherapy, but 5.9%±1.7% of cells obtained afterchemotherapy, formed mammospheres after 15 days of suspension culture(p<0.001, data not shown).

Similarly, the proportion of CD44⁺CD24⁻/low cells was 9.5-fold higher insamples after chemotherapy (p<0.001, Table 2). In another patient groupwith metastatic pleural effusions who had received chemotherapy 2-6years before, pleural cancer cells were highly enriched (31%±10%) forCD44⁺CD24⁻/low cells (data not shown). These data from three cohortssuggest that chemotherapy selectively enhances the proportionatesurvival of BT-IC.

TABLE 2 Incidence of tumors from primary breast cancer cells seriallytransplanted in NOD/SCID mice. 2 × 10³ cells 5 × 10³ cells PrimaryPrimary Number of cells inoculated Tumors Passage 1 Paggsate 2 TumorsPassage 1 Paggsate 2 lin⁻CD44⁺CD24^(−/low) Untransduced 6/8 8/8 8/8 8/88/8 8/8 Lentivirus 6/8 8/8 8/8 7/8 8/8 8/8 Lenti-let-7  2/8*   2/8^(∞)  2/8^(∞)   4/8^(∞)   4/8^(∞)  5/8* lin⁻NotCD44⁺CD24^(−/low) 0/8 0/8^(#)  0/8^(#)  0/8^(#) 0/8  0/8^(#) *= p < 0.05; ^(∞)= p < 0.01,^(#)= p < 0.001 compared with untransduced lin⁻CD44⁺CD24^(−/low) cells.For the initial inoculation, each mouse was inoculated with sortedcells, transduced or nor, from a different chemotherapy naïve humanpatient. For subsequent passages, cells were isolated, sorted, andtransduced forms from mice injected with tumor cells from the twopateients whos lenti-let7 transduced cells established xenographs.

By hematoxylin and eosin staining, the tissue structure and cellmorphology of the tumors generated from SKBR3, mammospheric SK-3rd orSK-3rd cells expressing let-7a were not grossly different (FIG. 4 b).However, H-RAS expression by immunohistochemistry was much higher inxenografts generated from mammosphere-derived SK-3rd cells as comparedto those from the parent SKBR3 cells. Transduction of SK-3rd withlet-7a-lentivirus, but not control lentivirus, significantly reducedH-RAS in the tumors, almost to the level of SKBR3-derived tumors (FIG. 4b,c). In keeping with the faster growth of the SK-3rd tumors, a higherproportion of SK-3rd-derived tumor cells than SKBR3-derived tumor cellsstained for the proliferating cell-associated antigen PCNA (FIG. 4 b,d).Transduction of SK-3rd with let7a-lentivirus also significantly reducedPCNA staining in the xenografted tumors, although not to that of theSKBR3-derived tumor. Therefore the inventors have demonstrated that thelack of let-7 plays an important role in the enhanced tumorigenicity andproliferation of tumor-initiating SK-3rd cells.

Example 9 Lack of Let-7 in Breast Tumor-Initiating Cells Promotes Lungand Liver Metastasis

It has been hypothesized that cancer cells migrate to distal sites toinitiate metastases only when they possess “stemness”^(14,15,34). Theinventors compared lung and liver metastases of the xenografts generatedfrom SK-3rd cells, expressing let-7a or not, and SKBR3 cells. Five weeksafter inoculation with 2×10⁵ mammospheric SK-3rd cells, massive lungmetastases were visualized by microscopy in 8 of 10 mice, but none ofthe mice injected with the same number of SKBR3 cells developedmicroscopic lung metastases within 9 weeks of inoculation (FIG. 5 a). Asan indicator of metastases, the wet lung weight of mice engrafted withSK-3rd was significantly higher (˜3-fold) than those injected withparental cells (P<0.01; FIG. 5 b). Transduction of SK-3rd withlenti-let-7 reduced both the numbers of mice with lung metastases from 8to 5 of 10 animals and the average lung weight by 44% (P<0.01). Themetastases were not only smaller, but also dispersed among alveoli (FIG.5 a), suggesting reduced clinical severity. The number of tumor cells inthe lung, quantified by qRT-PCR for human HPRT, was also 30% less in theanimals injected with let-7a-expressing SK-3rd compared with thoseinoculated with cells transduced with the empty vector (P<0.05; FIG. 5c).

Similarly, mammospheric SK-3rd cells developed micrometastases in thelivers of 6 of 10 mice inoculated with 2×10⁵ cells, but SKBR3 cells didnot (Table 1, FIG. 5 a). let-7a expression in SK-3^(rd) reduced both theoccurrence of liver metastasis by ˜50% as well as their size. This wasconfirmed by measuring a 58% reduction in human HPRT mRNA in the liversof animals inoculated with lenti-let-7-transduced cells (n=3) ascompared with those implanted with cells transduced with the controllentivirus (n=5, P<0.01; FIG. 5 c). Therefore, the lack of let-7 inbreast tumor-initiating cells contributes to their ability tometastasize to both the lung and liver.

The inventors have discovered breast tumors removed from patientstreated with preoperative chemotherapy are enriched for tumor-initiatingcells, also known as cancer stem cells. By taking advantage of thechemotherapeutic resistance of tumor-initiating cells, the inventorsgenerated a human breast cancer stem cell line (SK-3rd) by sequential invivo passage of a breast cancer cell line in immunodeficient micetreated with a low dose of a chemotherapeutic drug. The inventorsdiscovered that their generated cancer stem cell line, SK-3^(rd), hasmany of the hallmarks characteristic of stem cells, for instance,Sk-3^(rd) cells have the ability for self-renewal, multipotentdifferentiation and vigorous proliferative capacity, as well as theability to form mammospheres (or embryoid bodies), and are also positivefor breast cancer stem cell phenotype (Oct4⁺CD44⁺CD24⁻lineage)¹.Moreover, the inventors also discovered SK-3rd was much more malignantin immunodeficient mice than the parental line—it required 100-foldfewer cells to produce tumors, and surprisingly the tumors from theSK-3^(rd) metastasized, which did not occur at the same frequency in theparental line. Although there is a growing consensus that cancer stemcells are important in generating tumors and for resistance to therapyand metastasis, a major obstacle to their study is getting enough cellsbecause of their very low frequency in tumors^(9,10,12,37). Herein, theinventors have discovered a method for enriching for cancer stem cells,to enable unlimited numbers of cancer stem cells to be obtained. Themethods of the invention are useful for enriching for and obtainingunlimited numbers of cancer stem cells from any cancer type, for examplebreast cancer.

In vitro culture may produce epigenetic changes that might alter theproperties of tumor-initiating cells in subtle, not easily detectableways. Therefore, the inventors did as many of the studies as possiblewith freshly isolated in vivo passaged cells. Nonetheless, theself-renewing properties of the in vitro passaged mammospheres werehighly stable (FIG. 1 c), demonstrating that even with in vitro passage,mammospheres isolated from SK-3rd maintain “stemness” and theirself-renewal capacity. The inventors were careful to compare theirresults generated with an in vivo passaged cell line with those obtainedin freshly isolated primary breast cancer cells. The primary tumorsresected from adjuvant chemotherapy-treated patients had a surprisinglyhigh frequency (˜6%) of mammospheric cells with expected properties oftumor-initiating cells, a frequency that was only a third that of themouse-passaged stem cell line (16%) and 14 times that ofuntreated-patient tumors. Therefore, the inventors have discovered thatenriched primary breast tumor-initiating cells, or cancer stem cells,obtained from chemotherapy-treated patients are good source for studyingprimary tumor-initiating cells, particularly cancer stem cells fromhuman subject.

The inventors also discovered that the expression of let-7 miRNA andlet-7 homologues are significantly reduced or lacking intumor-initiating SK-3^(rd) cells, distinguishing SK-3^(rd)tumor-initiating cells from both their differentiated progeny and theparental cell line. Moreover, the inventors discovered that lack oflet-7 is required to maintain “stemness” and cancer stem cellsself-renewal capacity. By over-expressing let-7a in SK-3^(rd) cells, theinventors discovered that let-7 reduces self-renewal and proliferativecapability and converts highly malignant and metastasizingtumor-initiating into less malignant cells, similar to the parentalcells. Conversely, by antagonizing let-7 with antisense oligonucleotides(ASO) in the parental line, the inventors discovered that reduction oflet-7 in the parental line had the effect of enhancing its self-renewalpotential.

let-7 genes map to sites with frequent chromosomal instability duringoncogenesis⁴⁰, and let-7 is poorly expressed in lung^(24,33) and coloncancer⁴¹. Down-regulation of let-7 has not been reported in breastcancer⁴² let-7 has been postulated to work as a tumor suppressor gene bysilencing the expression of the RAS oncogenes³¹. The inventors alsodiscovered that in the SK-3^(rd) cancer stem cells which only expressHRAS, that the H-RAS protein, but not mRNA, was inversely correlatedwith let-7; H-RAS was high in tumor-initiating SK-3rd, but low indifferentiated SK-3rd and SKBR3. Moreover, the inventors discovered thatexogenous let-7a significantly knocked-down H-RAS. H-RAS is increased inup to 60% of human breast cancers^(43,44), but mutations arerare^(44,45). In additional experiments, the inventors discovered thatsilencing RAS to a level similar to that mediated by let-7 had much lessof an effect on cancer stem cell self-renewal and in vitrodifferentiation than over-expressing let-7, leading to the discoverythat RAS is not the only target of let-7 that contributes to maintaining“stemness” of cancer stem cells.

REFERENCES

The references cited herein and throughout the application areincorporated herein in their entirety by reference.

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1.-112. (canceled)
 113. A pharmaceutical composition comprising a let-7miRNA, a binding moiety and a targeting moiety, wherein the bindingmoiety connects the let-7 miRNA to the targeting moiety and wherein thetargeting moiety binds to the endothelial-specific marker(ESA)/Epithelial cell adhesion molecule (EpCAM) on the surface of acancer cell or cancer stem cell, and wherein the let-7 miRNA binds toand inhibits a RNA transcript comprising a let-7 target sequence. 114.The pharmaceutical composition of claim 113, wherein the let-7 targetsequence comprises SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO:11. 115.The pharmaceutical composition of claim 113, wherein the let-7 miRNA isselected from the group consisting of let-7a, let-7a1, let-7b, let-7c,let-7d, let-7e and let-7f.
 116. The pharmaceutical composition of claim113, wherein the miRNA is a pri-miRNA, pre-miRNA, mature miRNA effectivein gene silencing.
 117. The pharmaceutical composition of claim 113,wherein the let-7 miRNA comprises SEQ ID NO:1-8.
 118. The pharmaceuticalcomposition of claim 113, wherein the cancer is selected from at leastone of the group consisting of a pre-cancer, malignant cancer, therapyresistant cancer, breast cancer
 119. The pharmaceutical composition ofclaim 113, wherein the targeting moiety is selected from the groupconsisting of: an antibody, a single chain antibody, a Fab portion of anantibody and a (Fab′)2 segment.
 120. The pharmaceutical composition ofclaim 113, wherein the binding moiety is a protein or a nucleic acidbinding domain of a protein, and the binding moiety is fused to thecarboxyl terminus of the targeting moiety.
 121. The pharmaceuticalcomposition of claim 113, wherein the binding moiety is the proteinprotamine or nucleic acid binding fragment of protamine.
 122. A methodof treating or preventing cancer in a subject wherein the cancercomprises a cancer stem cell expressing the endothelial-specific marker(ESA)/Epithelial cell adhesion molecule (EpCAM), the method comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of: at least one let-7 miRNA agent; a binding moiety,wherein the binding moiety associates with the let-7 miRNA agent and thetargeting agent; and a targeting moiety, wherein the targeting moietybinds to the endothelial-specific marker (ESA)/Epithelial cell adhesionmolecule (EpCAM), wherein the let-7 miRNA binds to and inhibits a RNAtranscript comprising a let-7 target sequence which is expressed in thecancer stem cell, wherein inhibition of an RNA transcript comprising alet-7 target sequence inhibits proliferation of the cancer stem cell,thereby reducing or preventing the cancer in the subject.
 123. Themethod of claim 122, wherein the let-7 target sequence comprises SEQ IDNO: 9 or SEQ ID NO: 10 or SEQ ID NO:11.
 124. The method of claim 122,wherein the let-7 miRNA is selected from the group consisting of let-7a,let-7a1, let-7b, let-7c, let-7d, let-7e and let-7f.
 125. The method ofclaim 122, wherein the miRNA is a pri-miRNA, pre-miRNA, mature miRNAeffective in gene silencing.
 126. The method of claim 122, wherein thelet-7 miRNA comprises SEQ ID NO:1-8.
 127. The method of claim 21,wherein the cancer is selected from at least one of the group consistingof a pre-cancer, malignant cancer, therapy resistant cancer, breastcancer.
 128. The method of claim 122, wherein the targeting moiety isselected from the group consisting of: an antibody, a single chainantibody, a Fab portion of an antibody and a (Fab′)2 segment.
 129. Themethod of claim 122, wherein the binding moiety is a protein or anucleic acid binding domain of a protein, and the binding moiety isfused to the carboxyl terminus of the targeting moiety.
 130. The methodof claim 122, wherein the binding moiety is the protein protamine ornucleic acid binding fragment of protamine.
 131. The method of claim122, further comprising administering to the subject one or moreadditional cancer therapies selected from the group consisting ofsurgery, chemotherapy, radiotherapy, thermotherapy, immunotherapy,hormone therapy and laser therapy.
 132. The method of claim 122, whereinthe subject is a mammal or a human.